Novel compositions and processes for analyte detection, quantification and amplification

ABSTRACT

This invention provides novel compositions and processes for analyte detection, quantification and amplification. Nucleic acid arrays and libraries of analytes are usefully incorporated into such compositions and processes. Universal detection elements, signaling entities and the like are employed to detect and if necessary or desirable, to quantify analytes. Amplification of target analytes are also provided by the compositions and processes of this invention.

FIELD OF THE INVENTION

This invention relates to the field of analyte detection, quantificationand amplification, including compositions and processes directedthereto.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

The quantification of RNA expression provides major insights intoanalysis of cellular metabolism, function, growth and interactions.Although individual RNA species have historically been the subject ofthese studies, more interest is currently being shown in analysis of thepatterns of the simultaneous expression of multiple RNA species of bothknown and unknown function. This approach allows comparative studies onthe patterns of expression between different populations of cells,thereby serving as an indicator of the differences in biochemicalactivities taking place within these populations. For instance, a singlegroup of cells can be divided up into two or more populations where onegroup serves as a control and the other part is exposed to drugs,metabolites or different physical conditions. In this way, although themajority of the various species of mRNA show little or no differences inexpression levels, certain mRNA species may show dramatic increased ordecreased levels of expression compared to the untreated or normalcontrol.

As an example, it has long been known that the application of a phorbolester (PMA) results in changes in a large number of characteristics ofmammalian cells growing in vitro. In an experiment reported by Lockhartet al., (1996, Nature Biotechnology 14; 1675-1680) cells growing inculture were exposed to PMA and at various times afterwards, mRNA wasextracted and used to create a library of labeled probes. This materialwas subsequently hybridized to an array of nucleic acids that wascomplementary to various mRNA sequences. Significant changes could beseen in both the timing and the amount of induction of various cellularcytokines. On the other hand, so called “house-keeping” genes such asactin and GAPDH remained essentially unaffected by the treatment. Thisexample demonstrates that the various mRNA's can be independentlymonitored to determine which particular genes may be affected by atreatment.

Natural differences between cell populations can also be examined. Forinstance, differences in the expression levels of various genes can beobserved when cells progress through cell cycles (Cho et al., 1998 MolCell 2; 65-73 and Spellman et al., 1998 Mol. Biol. Cell 95;14863-14868). The gene expression profiles that were generated by thesestudies validated this approach when significant differences inexpression were observed for genes that had previously beencharacterized as encoding cell cycle related proteins. In addition, thearrays used in these studies comprised nucleic acid sequences thatrepresented the entire genetic complement of the yeast being studied. Assuch, one of the results of these studies was the observation of anumber of genes of previously unknown function that also displayed cellcycle dependent expression. Re-examination of these particular genes byother more conventional methods demonstrated that they were involved incell cycle progression. Thus, this method was demonstrated as beingcapable of recognizing genes previously known for differentialexpression and also for identifying new genes.

The differences between normal and transformed cells have also been asubject of long standing interest. The nature of the particular genesthat are either overexpressed or underexpressed relative to normal cellsmay provide information on the origination, progression or treatment ofcancerous cells. Array analysis has been carried out by using RNA fromtumor derived cells in comparison with expression from normal cells. Inone study by Perou et al (1999 Proc. Nat Acad. Sci. USA 96; 9212-9217)human mammary epithelial cells (HMEC) were compared with specimens fromprimary breast tumors. Included in this study were responses to variouscell factors as well as the results of confluence or senescence in thecontrol cultures. All of these are factors that may be involved oraffected by cellular transformation into the cancerous state. The amountof data generated in this type of study is almost overwhelming in itscomplexity. However distinct patterns or clusters of expression can beobserved that are correlated to factors associated with the specimens.Further understanding will also be gained when data is gathered fromexpression in other tumor types and their untransformed equivalents.

There are two distinct elements in all of the expression studies thatemploy arrays. The first element is concerned with the preparation ofthe bank of probes that will be used to bind or capture labeled materialthat is derived from the mRNAs that are being analyzed. The purpose ofthese arrays is to provide a multiplicity of individual probes whereeach probe is located in a discrete spatially defined position. Afterhybridization of the sample is carried out, the particular amount ofsample is measured for each site giving a relative measurement of howmuch material is present in the sample that has homology with theparticular probe that is located at that site. The two most commonlyused methods for array assembly operate on two very different scales forsynthesis of arrays.

On the simplest level of construction, discrete nucleic acids areaffixed to solid matrixes such as glass slides or nylon membranes in aprocess that is very similar to that employed by ink jet printers (Forexample, see Okamoto et al., 2000, Nature Biotechnology 18; 438-441).The nature of the probe deposited on the matrix can range from smallsynthetic oligonucleotides to large nucleic acid segments from clones.Preparation of a cloned segment to be used in this form of arrayassembly can range from E. coli colonies containing individual clonesthat are lysed and fixed directly onto a matrix or more elaborately byusing individual plasmids as templates for preparation of PCR amplifiedmaterial. The latter method is preferred due to the higher purity of thenucleic acid product. The choice of a particular probe to be used in theassembly can be directed in the sense that the function and sequence isknown. This of course will always be true when oligonucleotides are usedas the probes since they must be synthesized artificially. On the otherhand, when the probes are derived from larger cloned segments of DNA,they can be used irrespective of knowledge of sequence or function. Forinstance, a bank of probes that represent the entire yeast genome wasused in the studies cited earlier on differential expression during cellcycle progression. For human sequences, the burgeoning growth of thehuman sequencing project has provided a wealth of sequence informationthat is constantly expanding. Therefore, a popular source of probes thatcan be used to detect human transcripts has been Expressed Sequence Tags(ESTs) (Adams et al., 1991 Science 252; 1651-1656). The use of sequencesof unknown function has the advantage of a lack of any a prioriassumption concerning responsiveness in a comparative study and in fact,the study in itself may serve to identify functionality. At present,filter and glass arrays are commercially available from a number ofsources for the analysis of expression from various human tissues,developmental stages and disease conditions. On the other hand,directions for making custom arrays are widely disseminated throughoutthe literature and over the Internet.

At the other end of the scale in complexity is a process where in situsynthesis of oligonucleotides is carried out directly on a solid matrixusing a “masking” technology that is similar to that employed in etchingof microcircuits (Pirrung et al., U.S. Pat. No. 5,143,854, herebyincorporated by reference). Since this process can be carried out on avery small microscale, a very large number of different probes can beloaded onto a single “biochip” as a high density array. However, sincethis method depends upon site-specific synthesis, only oligonucleotidesare used and the probes are necessarily of limited size. Also, sincedirected sequence synthesis is used, sequence information has to beavailable for each probe. An advantage of this system is that instead ofa single probe for a particular gene product, a number of probes fromdifferent segments can be synthesized and incorporated into the designof the array. This provides a redundancy of information, establishingthat changes in levels of a particular transcript are due tofluctuations in the intended target rather than by transcripts with oneor more similar sequences. These “biochips” are commercially availableas well as the hardware and software required to read them.

Although solid supports such as plastic and glass have been commonlyused for fixation of nucleic acids, porous materials have also beenused. For example, oligonucleotides were joined to aldehyde groups inpolyacrylamide (Yershov et al., (1996) Proc Nat. Acad. Sci USA 93;4913-4918) and agarose (Afanassiev et al. (2000) Nucl. Acids Res. 28;e66) to synthesize arrays that were used in hybridization assays.

The second element involved in array analysis is the means by which thepresence and amount of labeled nucleic acids bound to the various probesof the array will be detected. There are three levels of use of thetarget mRNA that can provide signal generation. In the first approach,the native RNA itself can be labeled. This has been carried outenzymatically by phosphorylation of fragmented RNA followed by T4 RNAligase mediated addition of a biotinylated oligomer to the 5′ ends(Lockhart et al, 1996). This method has the limitation that it entailsan overnight incubation to insure adequate joining of labels to the RNA.For chemical labeling of RNA, the fragments can be labeled with psoralenthat has been linked to biotin (Lockhart et al, 1996). This method hasthe disadvantage that the crosslinking that joins the label to the RNAcan also lead to intrastrand crosslinking of target molecules reducingthe amount of hybridizable material.

In the second approach, rather than labeling the transcript itself, theRNA is used as a template to synthesize cDNA copies by the use of eitherrandom primers or by oligo dT primers. Extension of the primers byreverse transcriptase can be carried out in the presence of modifiednucleotides, thereby labeling all of the nascent cDNA copies. Themodified nucleotides can have moieties attached that generate signals inthemselves or they may have moieties suitable for attachment of othermoieties capable of generation of signals. Examples of groups that havebeen used for direct signal generation have been radioactive compoundsand fluorescent compounds such as fluorescein, Texas red, Cy3 and Cy 5.Direct signal generation has the advantage of simplicity but has thelimitation that in many cases there is reduced efficiency forincorporation of the labeled nucleotides by a polymerase. Examples ofgroups that have been used for indirect signal generation in arrays aredinitrophenol (DNP) or biotin ligands. Their presence is detected laterby the use of labeled molecules that have affinities for these ligands.Avidin or strepavidin specifically bind to biotin moieties andantibodies can be used that are specific for DNP or biotin. Theseproteins can be labeled themselves or serve as targets for secondarybindings with labeled compounds. Alternatively, when the labelednucleotides contain chemically active substituents such as allylaminemodifications, post-synthetic modification can be carried out by achemical addition of a suitably labeled ester.

The synthesis of a cDNA copy from an mRNA template essentially resultsin a one to one molar ratio of labeled product compared to startingmaterial. In some cases there may be limiting amounts of the mRNA beinganalyzed and for these cases, some amplification of the nucleic acidsequences in the sample may be desirable. This has led to the use of thethird approach, where the cDNA copy derived from the original mRNAtemplate is in itself used as a template for further synthesis. A systemtermed “Transcription Amplification System” (TAS) was described (Kwoh,D. Y. and Gingeras, T. R., 1989, Proc. Nat. Acad. Sci., 86, 1173-1177)in which a target specific oligonucleotide is used to generate a cDNAcopy and a second target specific oligonucleotide is used to convert thesingle stranded DNA into double-stranded form. By inclusion of a T7promoter sequence into the first oligonucleotide, the double-strandedmolecule can be used to make multiple transcription products that arecomplementary to the original mRNA of interest. The purpose of thissystem was for amplification of a discrete sequence from a pool ofvarious RNA species. No suggestion or appreciation of such a system forthe use of non-discrete primer sequences for general amplification wasdescribed in this work.

Multiple RNA transcript copies homologous to the original RNA populationhas been disclosed by van Gelder et al. in U.S. Pat. No. 5,891,636 wherespecific reference is given to the utility of such a system for creatinga library of various gene products in addition to discrete sequences.Since each individual mRNA molecule has the potential for ultimatelybeing the source of a large number of complementary transcripts, thissystem enjoys the advantages of linear amplification such that smalleramounts of starting material are necessary compared to direct labelingof the original mRNA or its cDNA copy.

However, the work described in U.S. Pat. No. 5,891,636 specificallyteaches away from addition of exogenous primers for synthesis of a2^(nd) strand. Instead, it discloses the use of oligonucleotide primersfor production of only the first strand of cDNA. For synthesis of thesecond strand, two possible methods were disclosed. In the first method,the nicking activity of RNase H on the original mRNA template was usedto create primers that could use the cDNA as a template. In the secondmethod, DNA polymerase was added to form hairpins at the end of thefirst cDNA strand that could provide self-priming. The first method hasa limitation that RNase H has to be added after the completion of thecDNA synthesis reaction and a balance of RNase H activity has to bedetermined to provide sufficient nicking without total degradation ofpotential RNA primers. The second method requires an extra step ofincubation a different polymerase besides the Reverse Transcriptase andalso S1 nuclease has to be added to eliminate the loop in the hairpinstructure. In addition, the formation and extension by foldback is apoorly understood system that does not operate at high efficiency wheresequences and amounts of cDNA copies may act as random factors.

In addition to the amplification provided by the use of RNAtranscription, PCR has been included in some protocols to carry outsynthesis of a library through the use of common primer binding sites ateach end of individual sequences (Endege et al., 1999 Biotechniques 26;542-550, Ying et al., 1999 Biotechniques 27; 410-414). These methodsshare the necessity for a machine dedicated to thermal cycling.

In addition to binding analytes from a library, the nucleic acids on anarray can use the analytes as templates for primer extension reactions.For instance, determination of Single Nucleotide Polymorphisms, (SNP's)has been carried out by the use of a set of primers at different siteson the array that exhibit sequence variations from each other (Pastinenet al., 2000, Genome Research 10; 1031-1042). The ability or inabilityof a template to be used for primer extension by each set of primers isan indication of the particular sequence variations within the analytes.More complex series of reactions have also been carried out by the useof arrays as platforms for localized amplification as described in U.S.Pat. No. 5,641,658 and Weslin et al., 2000, Nature Biotechnology 18;199-204. In these particular applications of array technology, PCR andSDA were carried out by providing a pair of unique primers for eachindividual nucleic acid target at each locus of the array. The presenceor absence of amplification at each locus of the array served as anindicator of the presence or absence of the corresponding targetsequences in the analyte samples.

Despite the accelerated development of the synthesis and use of DNAmicroarrays in recent years, the progress in the development of arraysof proteins or other ligands has been significantly slower even thoughsuch arrays are an ideal format with which to study gene expression, aswell as antibody-antigen, receptor-ligand, protein-protein interactionsand other applications. In previous art, protein arrays have been usedfor gene expression antibody screening, and enzymatic assays (Lueking etal. (1999) Anal. Biochem. 270; 103-111; de Wildt et al., (2000) NatureBiotechnology 18; 989-994, Arenkov et al., (2000) AnalyticalBiochemistry 278; 123-131). Protein arrays have also been used for highthroughput ELISA assays (Mendoza et al., (1999) Biotechniques 27;778-788) and for the detection of individual proteins in complexsolutions (Haab, et al.; (2001). Genome Biology 2; 1-13). However, theuse thus far has been limited because of the inherent problemsassociated with proteins. DNA is extremely robust and can, beimmobilized on a solid matrix, dried and rehydrated without any loss ofactivity or function. Proteins, however, are far more difficult toutilize in array formats. One of the main problems of using proteins inan array format is the difficulty of applying the protein to a solidmatrix in a form that would allow the protein to be accessible andreactive without denaturing or otherwise altering the peptide orprotein. Also, many proteins cannot be dehydrated and must be kept insolution at all times, creating further difficulties for use in arrays.

Some methods which have been used to prepare protein arrays includeplacing the proteins on a polyacrylamide gel matrix on a glass slidethat has been activated by treatment with glutaraldehyde or otherreagents (Arenkov, op. cit.). Another method has been the addition ofproteins to aldehyde coated glass slides, followed by blocking of theremaining aldehyde sites with BSA after the attachment of the desiredprotein. This method, however, could not be used for small proteinsbecause the BSA obscured the protein. Peptides and small proteins havebeen placed on slides by coating the slides with BSA and then activatingthe BSA with N,N′-disuccinimidyl carbonate (Taton et al., (2000) Science2789, 1760-1763). The peptides were then printed onto the slides and theremaining activated sites were blocked with glycine, Protein arrays havealso been prepared on poly-L-Lysine coated glass slides (Haab et al.,op. cit.) and agarose coated glass slides (Afanassiev et al., (2000)Nucleic Acids Research 28, e66). “Protein Chips” are also commerciallyavailable from Ciphergen (Fremont, Calif.) for a process where proteinsare captured onto solid surfaces and analyzed by mass spectroscopy.

The use of oligonucleotides as ‘hooks’ or ‘tags’ as identifiers fornon-nucleic acid molecules has been described in the literature. Forinstance, a library of peptides has been made where each peptide isattached to a discrete nucleic acid portion and members of the libraryare tested for their ability to bind to a particular analyte. Afterisolation of the peptides that have binding affinities, identificationwas carried out by PCR to “decode” the peptide sequence (Brenner. andLerner, (1992) Proc. Nat. Acad. Sci. USA 89; 5381-5383, Needels et al.,(1993) Proc. Nat. Acad. Sci. USA 90; 10,700-10,704). Nuceleic acidsequences have also been used as tags in arrays where selectedoligonucleotide sequences were added to primers used for singlenucleotide polymorphism genotyping (Hirschhorn, et al., (2000) Proc.Natl. Acad. Sc. USA, 97 12164-12169). However, in this case the ‘tag’ isactually part of the primer design and it is used specifically for SNPdetection using a single base extension assay. A patent applicationfiled by Lohse, et al., (WO 00/32823) has disclosed the use ofDNA-protein fusions for protein arrays. In this method, the protein issynthesized from RNA transcripts which are then reverse transcribed togive the DNA sequences attached to the corresponding protein. Thissystem lacks flexibility since the technology specifically relates onlyto chimeric molecules that comprise a nucleic acid and a peptide orprotein. In addition, the protein is directly derived from the RNAsequence so that the resultant DNA sequence is also dictated by theprotein sequence. Lastly, every protein that is to be used in an arrayrequires the use of an in vitro translation system made from cellextracts, a costly and inefficient system for large scale synthesis ofmultiple probes. The use of electrochemically addressed chips for usewith chimeric compositions has also been described by Bazin and Livache1999 in “Innovation and Perspectives in solid Phase Synthesis &Recombinatorial Libraries” R. Epton (Ed.) Mayflower Scientific Limited,Birmingham, UK.

SUMMARY OF THE INVENTION

This invention provides a composition of matter that comprises a libraryof analytes, the analytes being hybridized to an array of nucleic acids,the nucleic acids being fixed or immobilized to a solid support, whereinthe analytes comprise an inherent universal detection target (UDT), anda universal detection element (UDE) attached to the UDT, wherein the UDEgenerates a signal indicating the presence or quantity of the analytes,or the attachment of UDE to UDT.

This invention also provides a composition of matter that comprises alibrary of analytes, such analytes being hybridized to an array ofnucleic acids, and such nucleic acids being fixed or immobilized to asolid support, wherein the analytes comprise a non-inherent universaldetection target (UDT) and a universal detection element (UDE)hybridized to the UDT, and wherein the UDE generates a signal directlyor indirectly to detect the presence or quantity of such analytes.

The present invention further provides a composition of matter thatcomprises a library of analytes, such analytes being hybridized to anarray of nucleic acids, and such nucleic acids being fixed orimmobilized to a solid support, wherein the hybridization between theanalytes and the nucleic acids generate a domain for complex formation,and the composition further comprises a signaling entity complexed tothe domain.

The present invention yet further provides a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of: a) providing: (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified, whereineach of the nucleic acids of interest comprise at least one inherentuniversal detection target (UDT); and (iii) universal detection elements(UDE) which generates a signal directly or indirectly; b) hybridizingthe library (ii) with the array of nucleic acids (i) to form hybrids ifthe nucleic acids of interest are present; c) contacting the UDEs withthe UDTs to form a complex bound to the array; d) detecting orquantifying the more than one nucleic acid of interest by detecting ormeasuring the amount of signal generated from UDEs bound to the array.

Also provided by this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing: (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified, whereineach of the nucleic acids of interest comprise at least one inherentuniversal detection target (UDT); and (iii) universal detection elements(UDE) which generates a signal directly or indirectly; b) contacting theUDEs with the UDTs in the library of nucleic acid analytes to form oneor more complexes; c) hybridizing the library of nucleic acid analyteswith the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present; d) detecting or quantifying the more thanone nucleic acid of interest by detecting or measuring the amount ofsignal generated from UDEs bound to the array.

Also provided herein is a process for detecting or quantifying more thanone nucleic acid of interest in a library comprising the steps of a)providing (i) an array of fixed or immobilized nucleic acidscomplementary to the nucleic acids of interest; (ii) a library ofnucleic acid analytes which may contain the nucleic acids of interestsought to be detected or quantified, wherein each of the nucleic acidsof interest comprise at least one non-inherent universal detectiontarget (UDT), wherein the non-inherent UDT is attached to the nucleicacid analytes; and (iii) universal detection elements (UDE) whichgenerate a signal directly or indirectly; b) hybridizing the library(ii) with the array of nucleic acids (i) to form hybrids if the nucleicacids of interest are present; c) contacting the UDEs with the UDTs toform a complex bound to the array; d) detecting or quantifying the morethan one nucleic acid of interest by detecting or measuring the amountof signal generated from UDEs bound to the array.

Another aspect provided by this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified, whereineach of such nucleic acids of interest comprise at least onenon-inherent universal detection target (UDT), wherein the non-inherentUDTs are attached to the nucleic acid analytes; and (iii) universaldetection elements (UDE) which generate a signal directly or indirectly;b) contacting the UDEs with the UDTs in the library of nucleic acidanalytes to form one or more complexes; c) hybridizing the library (ii)with the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present; d) detecting or quantifying the more thanone nucleic acid of interest by detecting or measuring the amount ofsignal generated from UDEs bound to the array.

Another aspect provided by this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified; (iii)means for attaching one or more universal detection targets (UDT) to anucleic acid; (iv) universal detection elements (UDE) which generates asignal directly or indirectly; b) attaching such UDTs (iii) to thelibrary of nucleic acid analytes (ii); c) hybridizing the library (ii)with the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present; d) contacting the UDEs with the UDTs toform a complex bound to the array; e) detecting or quantifying the morethan one nucleic acid of interest by detecting or measuring the amountof signal generated from UDEs bound to the array.

Still another feature is process for detecting or quantifying more thanone nucleic acid of interest in a library comprising the steps of a)providing (i) an array of fixed or immobilized nucleic acidscomplementary to the nucleic acids of interest; (ii) a library ofnucleic acid analytes which may contain the nucleic acids of interestsought to be detected or quantified; (iii) means for attaching one ormore universal detection targets (UDT) to a nucleic acid; (iv) universaldetection elements (UDE) which generate a signal directly or indirectly;b) attaching the UDTs (iii) to the library of nucleic acid analytes(ii); c) contacting the UDEs with the UDTs in the library of nucleicacid analytes to form one or more complexes; d) hybridizing the library(ii) with the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present; e) detecting or quantifying the more thanone nucleic acid of interest by detecting or measuring the amount ofsignal generated from UDEs bound to the array.

The present invention provides additionally a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified; and (iii)universal detection elements (UDEs) which bind to a domain formed bynucleic acid hybrids for complex formation and generate a signaldirectly or indirectly; b) hybridizing the library (ii) with the arrayof nucleic acids (i) to form hybrids if such nucleic acids of interestare present, wherein any formed hybrids generate a domain for complexformation; c) contacting the UDEs with any hybrids to form a complexbound to the array; d) detecting or quantifying the more than onenucleic acid of interest by detecting or measuring the amount of signalgenerated from UDEs bound to the array.

Also provided herein is a composition of matter comprising a library offirst nucleic acid analyte copies, such first nucleic acid copies beinghybridized to an array of nucleic acids, those nucleic acids being fixedor immobilized to a solid support, wherein such first nucleic acidcopies comprise an inherent universal detection target (UDT) and auniversal detection element (UDE) attached to the UDT, wherein the UDEgenerates a signal directly or indirectly to detect the presence orquantity of any analytes.

Another embodiment of this invention is a composition of mattercomprising a library of first nucleic acid analyte copies, such firstnucleic acid copies being hybridized to an array of nucleic acids, thenucleic acids being fixed or immobilized to a solid support, whereinsuch first nucleic acid copies comprise one or more non-inherentuniversal detection targets (UDTs) and one or more universal detectionelements (UDEs) attached to the UDTs, wherein the UDEs generate a signaldirectly or indirectly to detect the presence or quantity of anyanalytes, and wherein the UDTs are either: (i) at the 5′ ends of thefirst nucleic acid copies and not adjacent to an oligoT segment orsequence, or (ii) at the 3′ ends of the first nucleic acid copies, or(iii) both (i) and (ii).

This invention also concerns a process for detecting or quantifying morethan one nucleic acid of interest in a library comprising the steps ofa) providing (i) an array of fixed or immobilized nucleic acidsidentical in part or whole to the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified, wherein each of suchnucleic acids of interest comprise at least one inherent universaldetection target (UDT); (iii) universal detection elements (UDE) whichgenerate a signal directly or indirectly; and (iv) polymerizing meansfor synthesizing nucleic acid copies of the nucleic acids of analytes;b) synthesizing one or more first nucleic acid copies which arecomplementary to all or part of the nucleic acid analytes andsynthesizing sequences which are complementary to all or part of the UDTto form a complementary UDT; c) hybridizing such first nucleic acidcopies with the array of nucleic acids (i) to form hybrids if suchnucleic acids of interest are present; d) contacting the UDEs with thecomplementary UDTs of the first nucleic acid copies to form a complexbound to the array; e) detecting or quantifying the more than onenucleic acid of interest by detecting or measuring the amount of signalgenerated from UDEs bound to the array.

Another embodiment provided by this invention is a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified, wherein each of such nucleic acids of interest comprise atleast one inherent universal detection target (UDT); (iii) universaldetection elements (UDE) which generate a signal directly or indirectly;and (iv) polymerizing means for synthesizing nucleic acid copies of suchnucleic acid analytes; b) synthesizing one or more first nucleic acidcopies of such nucleic acid analytes; c) contacting the UDEs with theUDTs in the first nucleic acid copies to form one or more complexes; d)hybridizing such first nucleic acid copies with the array of nucleicacids (i) to form hybrids if such nucleic acids of interest are present;and e) detecting or quantifying the more than one nucleic acid ofinterest by detecting or measuring the amount of signal generated fromUDEs bound to the array.

An additional aspect of the present invention is a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) means for attaching one or more non-inherent universaldetection targets (UDT) to a nucleic acid; (iv) universal detectionelements (UDE) which generate a signal directly or indirectly; and (v)polymerizing means for synthesizing nucleic acid copies of the nucleicacid analytes; b) attaching the non-inherent UDTs to either the 3′ endsof the nucleic acid analytes, the 5′ ends of the first nucleic acidanalytes, or both the 3′ ends and the 5′ ends of the nucleic acidanalytes; c) synthesizing one or more first nucleic acid copies of thenucleic acid analytes; d) hybridizing the first nucleic acid copies withthe array of nucleic acids (i) to form hybrids if such nucleic acids ofinterest are present; e) contacting the UDEs with the UDTs of the firstnucleic acid copies to form a complex bound to the array; f) detectingor quantifying the more than one nucleic acid of interest by detectingor measuring the amount of signal generated from UDEs bound to thearray.

Also provided herein is a process for detecting or quantifying more thanone nucleic acid of interest in a library comprising the steps of a)providing (i) an array of fixed or immobilized nucleic acids identicalin part or whole to the nucleic acids of interest; (ii) a library ofnucleic acid analytes which may contain the nucleic acids of interestsought to be detected or quantified; (iii) means for attaching one ormore non-inherent universal detection targets (UDT) to a nucleic acid;(iv) universal detection elements (UDE) which generated a signaldirectly or indirectly; and (v) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes; b) attaching suchnon-inherent UDTs to either the 3′ ends of the nucleic acid analytes,the 5′ ends of the first nucleic acid analytes, or both the 3′ ends andthe 5′ ends of the nucleic acid analytes; c) synthesizing one or morefirst nucleic acid copies of the nucleic acid analytes; d) contactingthe UDEs with the UDTs of the first nucleic acid copies to formcomplexes; e) hybridizing the first nucleic acid copies with the arrayof nucleic acids (i) to form hybrids if any nucleic acids of interestare present; f) detecting or quantifying the more than one nucleic acidof interest by detecting or measuring the amount of signal generatedfrom UDEs bound to the array.

Another embodiment provided herein is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to such nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) means for attaching one or more non-inherent universaldetection targets (UDT) to a nucleic acid; (iv) universal detectionelements (UDE) which generate a signal directly or indirectly; and (v)polymerizing means for synthesizing nucleic acid copies of the nucleicacid analytes; b) synthesizing one or more first nucleic acid copies ofthe nucleic acid analytes; c) attaching the non-inherent UDTs to eitherthe 3′ ends of the first nucleic acid copies, the 5′ ends of the firstnucleic acid copies, or both the 3′ ends and the 5′ ends of the firstnucleic acid copies; d) hybridizing the first nucleic acid copies withthe array of nucleic acids (i) to form hybrids if any nucleic acids ofinterest are present; e) contacting the UDEs with the UDTs of the firstnucleic acid copies to form a complex bound to the array; and f)detecting or quantifying the more than one nucleic acid of interest bydetecting or measuring the amount of signal generated from UDEs bound tothe array.

Another process provided by this invention is for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) means for attaching one or more non-inherent universaldetection targets (UDT) to a nucleic acid; (iv) universal detectionelements (UDE) which generate a signal directly or indirectly; and (v)polymerizing means for synthesizing nucleic acid copies of the nucleicacid analytes; b) synthesizing one or more first nucleic acid copies ofthe nucleic acid analytes; c) attaching the non-inherent UDTs to eitherthe 3′ ends of the first nucleic acid copies, the 5′ ends of the firstnucleic acid copies, or both the 3′ ends and the 5′ ends of the firstnucleic acid copies; d) contacting the UDEs with the UDTs of the firstnucleic acid copies to form a complex; e) hybridizing the first nucleicacid copies with the array of nucleic acids (i) to form hybrids if anynucleic acids of interest are present; and f) detecting or quantifyingthe more than one nucleic acid of interest by detecting or measuring theamount of signal generated from UDEs bound to the array.

Yet further provided is a process for detecting or quantifying more thanone nucleic acid of interest in a library comprising the steps of a)providing (i) an array of fixed or immobilized nucleic acidscomplementary to the nucleic acids of interest; (ii) a library ofnucleic acid analytes which may contain the nucleic acids of interestsought to be detected or quantified; (iii) universal detection elements(UDEs) which bind to a domain for complex formation formed by nucleicacid hybrids and generate a signal directly or indirectly; and (iv)polymerizing means for synthesizing nucleic acid copies of the nucleicacid analytes; b) synthesizing one or more nucleic acid copies of thenucleic acid analytes; c) hybridizing the first nucleic acid copies withthe array of nucleic acids (i) to form hybrids if any nucleic acids ofinterest are present, wherein any formed hybrids generate a domain forcomplex formation; d) contacting the UDEs with the hybrids to form acomplex bound to the array; and e) detecting or quantifying the morethan one nucleic acid of interest by detecting or measuring the amountof signal generated from UDEs bound to the array.

Another aspect provided by this invention is a composition of mattercomprising a library of double-stranded nucleic acids substantiallyincapable of in vivo replication and free of non-inherent homopolymericsequences, the nucleic acids comprising sequences complementary oridentical in part or whole to inherent sequences of a library obtainedfrom a sample, wherein the double-stranded nucleic acids comprise atleast one inherent universal detection target (UDT) proximate to one endof the double strand and at least one non-inherent production centerproximate to the other end of the double strand.

Yet another aspect of this invention concerns a composition of mattercomprising a library of double-stranded nucleic acids substantiallyincapable of in vivo replication, such nucleic acids comprisingsequences complementary or identical in part or whole to inherentsequences of a library obtained from a sample, wherein thedouble-stranded nucleic acids comprise at least four (4) non-inherentnucleotides proximate to one end of the double strand and a non-inherentproduction center proximate to the other end of the double strand.

Among other useful aspects of this invention is a composition of mattercomprising a library of double-stranded nucleic acids fixed to a solidsupport, those nucleic acids comprising sequences complementary oridentical in part or whole to inherent sequences of a library obtainedfrom a sample and the nucleic acids further comprising at least onefirst sequence segment of non-inherent nucleotides proximate to one endof the double strand and at least one second sequence segment proximateto the other end of the double strand, the second sequence segmentcomprising at least one production center.

Another feature of this invention is a composition of matter comprisinga library of double-stranded nucleic acids attached to a solid support,the nucleic acids comprising sequences complementary or identical inpart or whole to inherent sequences of a library obtained from a sample,wherein the double-stranded nucleic acids comprise at least one inherentuniversal detection target (UDT) proximate to one end of the doublestrand and at least one non-inherent production center proximate to theother end of the double strand.

The invention herein also provides a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, the polymerizing meanscomprising a first set of primers and a second set of primers, whereinthe second set of primers comprises at least two segments, the firstsegment at the 3′ end comprising random sequences, and the secondsegment comprising at least one production center; (iv) means forsynthesizing nucleic acid copies under isothermal or isostaticconditions; b) contacting the library of nucleic acid analytes with thefirst set of primers to form more than one first bound entity; c)extending the bound first set of primers by means of template sequencesprovided by the nucleic acid analytes to form first copies of theanalytes; d) contacting the extended first copies with the second set ofprimers to form more than one second bound entity; e) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; f)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; g) hybridizing any nucleic acid copies formed instep f) to the array of nucleic acids provided in step a) (i); and h)detecting or quantifying any of the hybridized copies obtained in stepg).

Also provided by this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers comprise at least one productioncenter; and (iv) means for synthesizing nucleic acid copies underisothermal or isostatic conditions; b) contacting the library of nucleicacid analytes with the first set of primers to form more than one firstbound entity; c) extending the bound first set of primers by means oftemplate sequences provided by the nucleic acid analytes to form firstcopies of the analytes; d) extending the first copies by means of atleast four (4) or more non-inherent homopolymeric nucleotides; e)contacting the extended first copies with the second set of primers toform more than one second bound entity; f) extending the bound secondset of primers by means of template sequences provided by the extendedfirst copies to form more than one complex comprising extended firstcopies and extended second set of primers; g) synthesizing from aproduction center in the second set of primers in the complexes one ormore nucleic acid copies under isothermal or isostatic conditions; h)hybridizing the nucleic acid copies formed in step g) to the array ofnucleic acids provided in step a) (i); and i) detecting or quantifyingany of the hybridized copies obtained in step h).

Another feature of this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set comprises at least one production center; (iv) aset of oligonucleotides or polynucleotides complementary to at least onesegment or sequence of the second set of primers; and (v) means forligating the set of oligonucleotides or polynucleotides (iv); b)contacting the library of nucleic acid analytes with the first set ofprimers to form more than one first bound entity; c) extending the boundfirst set of primers by means of template sequences provided by thenucleic acid analytes to form first copies of the analytes; d) ligatingthe set of oligonucleotides or polynucleotides a) (iv) to the 3′ end ofthe first copies formed in step c) to form more than one ligatedproduct; e) contacting the ligated product with the second set ofprimers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph).

Still yet further this invention provides a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the second set comprises at least one production center; (iv) aset of oligonucleotides or polynucleotides complementary to at least onesegment or sequence of the second set of primers; and (v) means forligating the set of oligonucleotides or polynucleotides (iv); b)contacting the library of nucleic acid analytes with the first set ofprimers to form more than one first bound entity; c) extending the boundfirst set of primers by means of template sequences provided by thenucleic acid analytes to form first copies of the analytes; d) ligatingthe set of oligonucleotides or polynucleotides a) (iv) to the 3′ end ofthe first copies formed in step c) to form more than one ligatedproduct; e) contacting the ligated product with the second set ofprimers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph).

Still yet further provided by this invention is a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers, a second set of primers and athird set of primers wherein the third set comprises at least oneproduction center; and b) contacting the library of nucleic acidanalytes with the first set of primers to form a first set of boundprimers; c) extending the first set of bound primers by means oftemplate sequences provided by the nucleic acid analytes to form firstcopies of the analytes; d) contacting the extended first copies with thesecond set of primers to form a second set of bound primers; e)extending the second set of bound primers by means of template sequencesprovided by the extended first copies to form second copies of thenucleic acid analytes; f) contacting the second copies with the thirdset of primers to form more than one third bound entity to form a thirdset of bound primers; g) extending the third set of bound primers bymeans of template sequences provided by the extended second set ofprimers to form a hybrid comprising a second copy, a third copy and atleast one production center; h) synthesizing from the production centerin the second set of primers in the complexes one or more nucleic acidcopies under isothermal or isostatic conditions; i) hybridizing thenucleic acid copies formed in step i) to the array of nucleic acidsprovided in step a) (i); and j) detecting or quantifying any of thehybridized copies obtained in step i).

Also uniquely provided in this invention is a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers are fixed or immobilized to a solidsupport, and wherein the second set of primers comprises at least twosegments, the first segment at the 3′ end comprising random sequences,and the second segment comprising at least one production center; (iv)means for synthesizing nucleic acid copies under isothermal or isostaticconditions; b) contacting the library of nucleic acid analytes with thefirst set of primers to form more than one first bound entity; c)extending the bound first set of primers by means of template sequencesprovided by the nucleic acid analytes to form first copies of theanalytes; d) contacting the extended first copies with the second set ofprimers to form more than one second bound entity; e) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; f)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; g) hybridizing the nucleic acid copies formed instep f) to the array of nucleic acids provided in step a) (i); and h)detecting or quantifying any of the hybridized copies obtained in stepg).

Another significant aspect of this invention is a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers are fixed or immobilized to a solidsupport, and wherein the first set of primers comprise at least oneproduction center; and (iv) means for synthesizing nucleic acid copiesunder isothermal or isostatic conditions; b) contacting the library ofnucleic acid analytes with the first set of primers to form more thanone first bound entity; c) extending the bound first set of primers bymeans of template sequences provided by the nucleic acid analytes toform first copies of the analytes; d) extending the first copies bymeans of at least four (4) or more non-inherent homopolymericnucleotides; e) contacting the extended first copies with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; g)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph).

Also provided in accordance with the present invention is a process fordetecting or quantifying more than one nucleic acid of interest in alibrary comprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers are fixed or immobilized to a solidsupport, and wherein the first set comprises at least one productioncenter; (iv) a set of oligonucleotides or polynucleotides complementaryto at least one segment or sequence of the second set of primers; and(v) means for ligating the set of oligonucleotides or polynucleotides(iv); b) contacting the library of nucleic acid analytes with the firstset of primers to form more than one first bound entity; c) extendingthe bound first set of primers by means of template sequences providedby the nucleic acid analytes to form first copies of the analytes; d)ligating the set of oligonucleotides or polynucleotides a) (iv) to the3′ end of the first copies formed in step c) to form more than oneligated product; e) contacting the ligated product with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph).

Another feature of the present invention concerns a process fordetecting or quantifying more than one nucleic acid of interest in alibrary comprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers are fixed or immobilized to a solidsupport, and wherein the second set comprises at least one productioncenter; (iv) a set of oligonucleotides or polynucleotides complementaryto at least one segment or sequence of the second set of primers; and(v) means for ligating the set of oligonucleotides or polynucleotides(iv); b) contacting the library of nucleic acid analytes with the firstset of primers to form more than one first bound entity; c) extendingthe bound first set of primers by means of template sequences providedby the nucleic acid analytes to form first copies of the analytes; d)ligating the set of oligonucleotides or polynucleotides a) (iv) to the3′ end of the first copies formed in step c) to form more than oneligated product; e) contacting the ligated product with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph).

Yet another process is provided by this invention, the process being onefor detecting or quantifying more than one nucleic acid of interest in alibrary and comprising the steps of a) providing (i) an array of fixedor immobilized nucleic acids identical or complementary in part or wholeto sequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers, a second set of primers and athird set of primers, wherein the first set of primers are fixed orimmobilized to a solid support, and wherein the third set comprises atleast one production center; and b) contacting the library of nucleicacid analytes with the first set of primers to form more than one firstbound entity; c) extending the bound first set of primers by means oftemplate sequences provided by the nucleic acid analytes to form firstcopies of the analytes; d) contacting the extended first copies with thesecond set of primers to form more than one second bound entity; e)extending the bound second set of primers by means of template sequencesprovided by the extended first copies to form an extended second set ofprimers; f) separating the extended second set of primers obtained instep e); g) contacting the extended second set of primers with the thirdset of primers to form more than one third bound entity; h) extendingthe third bound entity by means of template sequences provided by theextended second set of primers to form more than one complex comprisingthe extended third bound entity and the extended set of primers; i)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; j) hybridizing the nucleic acid copies formed instep i) to the array of nucleic acids provided in step a) (i); and k)detecting or quantifying any of the hybridized copies obtained in stepj).

Another significant embodiment provided herein is a process fordetecting or quantifying more than one nucleic acid of interest in alibrary comprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to sequences of thenucleic acids of interest; (ii) a library of nucleic acid analytes whichmay contain the nucleic acids of interest sought to be detected orquantified; and (iii) polymerizing means for synthesizing nucleic acidcopies of the nucleic acid analytes, such polymerizing means comprisinga first set of primers; b) contacting the nucleic acid analytes with thefirst set of primers to form a first bound entity; c) extending thebound set of first set of primers by means of template sequencesprovided by the nucleic acid analytes to form first nucleic acid copiesof the analytes; d) separating the first nucleic acid copies from theanalytes; e) repeating steps b), c) and d) until a desirable amount offirst nucleic acid copies have been synthesized; f) hybridizing thenucleic nucleic acid copies formed in step e) to the array of nucleicacids provided in step (i); and g) detecting or quantifying any of thehybridized first nucleic acid copies obtained in step f).

The invention described herein also provides a process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to sequences of thenucleic acids of interest; (ii) a library of nucleic acid analytes whichmay contain the nucleic acids of interest sought to be detected orquantified; (iii) polymerizing means for synthesizing nucleic acidcopies of the nucleic acid analytes, such polymerizing means comprisinga first set of primers and a second set of primers; (iv) means foraddition of sequences to the 3′ end of nucleic acids; b) contacting thenucleic acid analytes with the first set of primer to form a first boundentity; c) extending the bound set of first set of primers by means oftemplate sequences provided by the nucleic acid analytes to form firstnucleic acid copies of the analytes; d) extending the first nucleiccopies by the addition of non-template derived sequences to the 3′ endof the first nucleic acid copies; e) contacting the extended firstnucleic acid copies with the second set of primers to form a secondbound entity; f) extending the bound set of second set of primers bymeans of template sequences provided by the extended first nucleic acidcopies to form second nucleic acid copies; g) separating the secondnucleic acid copies from the extended first nucleic acid copies; h)repeating steps e), f) and g) until a desirable amount of second nucleicacid copies have been synthesized; i) hybridizing the second nucleicacid copies formed in step h) to the array of nucleic acids provided instep (i); and j) detecting or quantifying any of the hybridized secondnucleic acid copies obtained in step i).

Among other significant compositions provided by the present inventionis a composition of matter that comprises an array of solid surfacescomprising discrete areas, wherein at least two of the discrete areaseach comprises a first set of nucleic acid primers; and a second set ofnucleic acid primers; wherein the nucleotide sequences in the first setof nucleic acid primers are different from the nucleotide sequences inthe second set of nucleic acid primers; wherein the nucleotide sequencesof a first set of nucleic acid primers of a first discrete area and thenucleotide sequences of a first set of nucleic acid primers of a seconddiscrete area differ from each other by at least one base; and whereinthe nucleotide sequences of the second set of nucleic acid primers of afirst discrete area and the nucleotide sequences of the second set ofnucleic acid primers of a second discrete area are substantially thesame or identical.

A related composition of this invention concerns a composition of matterthat comprises an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of the discrete areas eachcomprises a first set of nucleic acid primers; and a second set ofnucleic acid primers; wherein the nucleotide sequences in the first setof nucleic acid primers are different from the nucleotide sequences inthe second set of nucleic acid primers; wherein the nucleotide sequencesof a first set of nucleic acid primers of a first discrete area and thenucleotide sequences of a first set of nucleic acid primers of a seconddiscrete area differ substantially from each other; and wherein thenucleotide sequences of the second set of nucleic acid primers of afirst discrete area and the nucleotide sequences of the second set ofnucleic acid primers of a second discrete area are substantially thesame or identical.

Related to the last-mentioned compositions are processes for producingtwo or more copies of nucleic acids of interest in a library comprisingthe steps of a) providing (i) an array of solid surfaces comprising aplurality of discrete areas; wherein at least two of the discrete areaseach comprises: (1) a first set of nucleic acid primers; and (2) asecond set of nucleic acid primers; wherein the nucleotide sequences inthe first set of nucleic acid primers are different from the nucleotidesequences in the second set of nucleic acid primers; wherein thenucleotide sequences of a first set of nucleic acid primers of a firstdiscrete area and the nucleotide sequences of a first set of nucleicacid primers of a second discrete area differ from each other by atleast one base; and wherein the nucleotide sequences of the second setof nucleic acid primers of a first discrete area and the nucleotidesequences of the second set of nucleic acid primers of a second discretearea are substantially the same or identical; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest; (iii)polymerizing means for synthesizing nucleic acid copies of the nucleicacids of interest; b) contacting a primer of the first set with acomplementary sequence in the nucleic acid of interest; c) extending theprimer in the first set using the nucleic acid of interest as a templateto generate an extended first primer; d) contacting a primer in thesecond set with a complementary sequence in the extended first primer;e) extending the primer in the second set using the extended firstprimer as a template to generate an extended second primer; f)contacting a primer in the first set with a complementary sequence inthe extended second primer; g) extending the primer in the first setusing the extended second primer as a template to generate an extendedfirst primer; and h) repeating steps d) through g) above one or moretimes.

Another related process of the present invention is useful for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of suchdiscrete areas each comprises: (1) a first set of nucleic acid primers;and (2) a second set of nucleic acid primers; wherein the nucleotidesequences in the first set of nucleic acid primers are different fromthe nucleotide sequences in the second set of nucleic acid primers;wherein the nucleotide sequences of a first set of nucleic acid primersof a first discrete area and the nucleotide sequences of a first set ofnucleic acid primers of a second discrete area differ from each other byat least one base; and wherein the nucleotide sequences of the secondset of nucleic acid primers of a first discrete area and the nucleotidesequences of the second set of nucleic acid primers of a second discretearea are substantially the same or identical; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest; (iii)polymerizing means for synthesizing nucleic acid copies of the nucleicacids of interest; and (iv) non-radioactive signal generating meanscapable of being attached to or incorporated into nucleic acids; b)contacting a primer of the first set with a complementary sequence inthe nucleic acid of interest; c) extending the primer in the first setusing the nucleic acid of interest as a template to generate an extendedfirst primer; d) contacting a primer in the second set with acomplementary sequence in the extended first primer; e) extending theprimer in the second set using the extended first primer as a templateto generate an extended second primer; f) contacting a primer in thefirst set with a complementary sequence in the extended second primer;g) extending the primer in the first set using the extended secondprimer as a template to generate an extended first primer; h) repeatingsteps d) through g) above one or more times; and i) detecting orquantifying by means of the non-radioactive signal generating meansattached to or incorporated into any of the extended primers in stepsc), e), g), and h).

Another useful composition provided by the present invention is acomposition of matter that comprises an array of solid surfacescomprising a plurality of discrete areas, wherein at least two of suchdiscrete areas comprise: a chimeric composition comprising a nucleicacid portion; and a non-nucleic acid portion, wherein the nucleic acidportion of a first discrete area has the same sequence as the nucleicacid portion of a second discrete area, and wherein the non-nucleic acidportion has a binding affinity for analytes of interest.

Further provided by the present invention is a composition of matterthat comprises an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of the discrete areas comprise achimeric composition hybridized to complementary sequences of nucleicacids fixed or immobilized to the discrete areas, wherein the chimericcomposition comprises a nucleic acid portion, and a non-nucleic acidportion, the nucleic acid portion comprising at least one sequence,wherein the non-nucleic acid portion has a binding affinity for analytesof interest, and wherein when the non-nucleic acid portion is a peptideor protein, the nucleic acid portion does not comprises sequences whichare either identical or complementary to sequences that code for suchpeptide or protein.

Also provided as a significant aspect of the present invention is aprocess for detecting or quantifying analytes of interest, the processcomprising the steps of 1) providing a) an array of solid surfacescomprising a plurality of discrete areas, wherein at least two of suchdiscrete areas comprise a chimeric composition comprising a nucleic acidportion, and a non-nucleic acid portion; wherein the nucleic acidportion of a first discrete area has the same sequence as the nucleicacid portion of a second discrete area; and wherein the non-nucleic acidportion has a binding affinity for analytes of interest; b) a samplecontaining or suspected of containing one or more of the analytes ofinterest; and c) signal generating means; 2) contacting the array a)with the sample b) under conditions permissive of binding the analytesto the non-nucleic acid portion; 3) contacting the bound analytes withthe signal generating means; and 4) detecting or quantifying thepresence of the analytes.

Another feature provided by the present invention is a process fordetecting or quantifying analytes of interest, this process comprisingthe steps of 1) providing a) an array of solid surfaces comprising aplurality of discrete areas; wherein at least two of such discrete areascomprise a chimeric composition comprising a nucleic acid portion; and anon-nucleic acid portion; wherein the nucleic acid portion of a firstdiscrete area has the same sequence as the nucleic acid portion of asecond discrete area; and wherein the non-nucleic acid portion has abinding affinity for analytes of interest; b) a sample containing orsuspected of containing one or more of the analytes of interest; and c)signal generating means; 2) labeling the analytes of interest with thesignal generating means; 3) contacting the array a) with the labeledanalytes under conditions permissive of binding the labeled analytes tothe non-nucleic acid portion; and 4) detecting or quantifying thepresence of the analytes.

Also provided by the present invention is a process for detecting orquantifying analytes of interest, the process comprising the steps of 1)providing a) an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of such discrete areas comprisenucleic acids fixed or immobilized to such discrete areas, b) chimericcompositions comprising: i) a nucleic acid portion; and ii) anon-nucleic acid portion; the nucleic acid portion comprising at leastone sequence, wherein the non-nucleic acid portion has a bindingaffinity for analytes of interest, and wherein when the non-nucleic acidportion is a peptide or protein, the nucleic acid portion does notcomprise sequences which are either identical or complementary tosequences that code for the peptide or protein; c) a sample containingor suspected of containing the analytes of interest; and d) signalgenerating means; 2) contacting the array with the chimeric compositionsto hybridize the nucleic acid portions of the chimeric compositions tocomplementary nucleic acids fixed or immobilized to the array; 3)contacting the array a) with the sample b) under conditions permissiveof binding the analytes to the non-nucleic acid portion; 4) contactingthe bound analytes with the signal generating means; and 5) detecting orquantifying the presence of the analytes.

Additionally this invention provides a process for detecting orquantifying analytes of interest, the process comprising the steps of 1)providing a) an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of the discrete areas comprisenucleic acids fixed or immobilized to the discrete areas, b) chimericcompositions comprising i) a nucleic acid portion; and ii) a non-nucleicacid portion, the nucleic acid portion comprising at least one sequence,wherein the non-nucleic acid portion has a binding affinity for analytesof interest, and wherein when the non-nucleic acid portion is a peptideor protein, the nucleic acid portion does not comprise sequences whichare either identical or complementary to sequences that code for thepeptide or protein; c) a sample containing or suspected of containingthe analytes of interest; and d) signal generating means; 2) contactingthe chimeric compositions with the sample b) under conditions permissiveof binding the analytes to the non-nucleic acid portion; 3) contactingthe array with the chimeric compositions to hybridize the nucleic acidportions of the chimeric compositions to complementary nucleic acidsfixed or immobilized to the array; 4) contacting the bound analytes withthe signal generating means; and 5) detecting or quantifying thepresence of the analytes.

Another useful provision of the invention herein is a process fordetecting or quantifying analytes of interest, such process comprisingthe steps of 1) providing a) an array of solid surfaces comprising aplurality of discrete areas; wherein at least two of the discrete areascomprise nucleic acids fixed or immobilized to the discrete areas, b)chimeric compositions comprising i) a nucleic acid portion; and ii) anon-nucleic acid portion; the nucleic acid portion comprising at leastone sequence, wherein the non-nucleic acid portion has a bindingaffinity for analytes of interest, and wherein when the non-nucleic acidportion is a peptide or protein, the nucleic acid portion does notcomprise sequences which are either identical or complementary tosequences that code for the peptide or protein; c) a sample containingor suspected of containing the analytes of interest; and d) signalgenerating means; 2) contacting the array with the chimeric compositionsto hybridize the nucleic acid portions of the chimeric compositions tocomplementary nucleic acids fixed or immobilized to the array; 3)labeling the analytes of interest with the signal generating means; 4)contacting the array with the labeled analytes to bind the analytes tothe non-nucleic acid portion; and 5) detecting or quantifying thepresence of the analytes.

Yet further provided by the present invention is a process for detectingor quantifying analytes of interest, the process comprising the stepsof 1) providing a) an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of the discrete areas comprisenucleic acids-fixed or immobilized to the discrete areas, b) chimericcompositions comprising: i) a nucleic acid portion; and ii) anon-nucleic acid portion; the nucleic acid portion comprising at leastone sequence, wherein the non-nucleic acid portion has a bindingaffinity for analytes of interest, and wherein when the non-nucleic acidportion is a peptide or protein, such nucleic acid portion does notcomprise sequences which are either identical or complementary tosequences that code for the peptide or protein; c) a sample containingor suspected of containing the analytes of interest; and d) signalgenerating means; 2) contacting the array with the chimeric compositionsto hybridize the nucleic acid portions of the chimeric compositions tocomplementary nucleic acids fixed or immobilized to the array; 3)labeling the analytes of interest with the signal generating means; 4)contacting the array with the labeled analytes to bind the analytes tothe non-nucleic acid portion; and 5) detecting or quantifying thepresence of the analytes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an array with mRNA from a library of analytes with UDTs.

FIG. 2 shows fragmentation of analytes followed by addition ofnon-inherent UDTs to analytes.

FIG. 3 depicts the incorporation of a non-inherent UDT to a 1st cNA copyby means of a primer.

FIG. 4 illustrates the use of Random Primers with Production Centers for2^(nd) strand synthesis.

FIG. 5 relates to the same process as FIG. 4 wherein the ProductionCenters are double-stranded.

FIG. 6 illustrates 2nd cNA strand priming at terminal and internalsites.

FIG. 7 illustrates 2nd cNA strand priming after Terminal transferaseaddition of homopolymeric sequences.

FIG. 8 shows the addition of primer binding sites by ligation.

FIG. 9 illustrates multiple additions of primer binding sites.

FIG. 10 shows 1st strand synthesis by extension of an oligo dT primerbound to a bead followed by 2nd cNA strand synthesis with random primershaving production centers.

FIG. 11 illustrates 1st strand synthesis from poly T primer indirectlybound to a bead followed by 2nd strand synthesis with random primershaving production center.

FIG. 12 shows the incorporation of a promoter during 3rd strandsynthesis.

FIG. 13 illustrates the synthesis of an amplicon for isothermalamplification of a library of analytes.

FIG. 14 shows the synthesis of an amplicon for SDA amplification.

FIG. 15 shows the ligation of a primer binding site for isothermalamplification.

FIG. 16 shows the binding of an analyte to an array with SPEs and UPEsfor solid phase amplification.

FIG. 17 shows the extension of an SPE on an array during solid phaseamplification.

FIG. 18 shows the binding of an UPE to an extended SPE followed byextension of the UPE during solid phase amplification.

FIG. 19 shows solid phase amplification in which binding of extendedSPEs and UPEs to unextended SPEs and UPEs occur.

FIG. 20 depicts an amplification array for comparative analysis.

FIG. 21 illustrates the use of an array with SPEs and UPEs for SNPanalysis.

FIG. 22 relates to binding of analytes to SPEs on an array.

FIG. 23 shows the binding of primers to extended SPEs on an array.

FIG. 24 demonstrates the binding of primers and extended primers to SPEson an array.

FIG. 25 shows the extension of primers and SPEs on an array inaccordance with amplification disclosed in this invention.

FIG. 26 depicts the binding of nucleic acid portions of chimericcompositions to complementary sequences on an array

FIG. 27 is a gel analysis illustrating the dependency on ReverseTranscriptase for the amplification of a library in accordance with thisinvention and Example 3 below.

FIG. 28 is a gel analysis that demonstrates transcription after multiplerounds of 2nd strand synthesis as described further below in Example 4.

FIG. 29 is also a gel analysis that shows second round of RNAtranscription from a library as described in Example 5 below.

FIG. 30 is a gel analysis also shows transcription from library madeafter poly dG tailing in accordance with the present invention andExample 6 below.

FIG. 31 is a gel analysis that shows RNA transcription after a series ofreactions one of which was 2nd strand synthesis by thermostable DNApolymerases as described in Example 9 below.

FIG. 32 is a gel analysis that shows transcription from libraries madefrom sequential synthesis of 2nd strands as further described in Example10 below.

FIG. 33 is also a gel analysis of amplification of a library of analytesusing various reverse transcriptases for 1st stand synthesis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel methods, compositions and kitsthat can be used in making and analyzing a library of nucleic acids. Thenucleic acids in the sample being tested can be used directly for signalgeneration or they can be used as templates to provide one or morenucleic acid copies that comprise sequences that are either identical orcomplementary to the original sequences.

In the present invention the following terms are used and defined below:

An analyte is a biological polymer or ligand that is isolated or derivedfrom biological sources such as organs, tissues or cells, ornon-biological sources by synthetic or enzymatic means or processes.Examples of biological polymers can include but are not limited tooligonucleotides, polynucleotides, oligopeptides, polypeptides,oligosaccharides, polysaccharides and lipids. Examples of ligands caninclude but are not necessarily limited to non-peptide antigens,hormones, enzyme substrates, vitamins, drugs, and non-peptide signalmolecules.

A library is a diverse collection of nucleic acids that comprises: a)analytes; b) nucleic acids derived from analytes that comprise sequencesthat are complementary to sequences in the analytes; c) nucleic acidsderived from analytes that comprise sequences that are identical tosequences in the analytes; and d) any combination of the foregoing.

A label is any moiety that is capable of directly or indirectlygenerating a signal.

A production center is a segment of a nucleic acid or analogue thereofthat is capable of producing more than one copy of a sequence that isidentical or complementary to sequences that are operably linked to theproduction center.

Universal Detection Targets (UDTs) are defined as common or conservedsegments in diverse nucleic acids that are present in populations ofnucleic acids in a sample and are capable of recognition by acorresponding binding partner. The UDTs may be intrinsic or they may beartificially incorporated into nucleic acids. Examples of inherent UDTscan comprise but not be limited to 3′ poly A segments, 5′ caps,secondary structures and consensus sequences. Examples of inherentconsensus sequences that might find use in the present invention cancomprise but not be limited to signal sites for poly A addition,splicing elements and multicopy repeats such as Alu sequences. UDTs mayalso be artificially incorporated into nucleic acids by an addition tothe original analyte nucleic acid or during synthesis of nucleic acidsthat comprise sequences that are identical or complementary to thesequences of the original analytes. Artificially added UDTs may belabeled themselves or they may serve as binding partners.

Universal Detection Elements (UDEs) are comprised of two segments: afirst segment that is capable of acting as a binding partner for a UDTand a second segment that is either labeled or otherwise capable ofgenerating a detectable signal. In some cases the first and secondsegments can be overlapping or even comprise the same segments. WhenUDEs are labeled, they may comprise a single signal moiety or they maycomprise more than one signal entity. Segments of UDEs involved inbinding to UDTs or signal generation may comprise but not be limited topolymeric substances such as nucleic acids, nucleic acid analogues,polypeptides, polysacharides or synthetic polymers.

The present invention discloses the use of UDTs and UDEs for the purposeof array analysis. The present invention also discloses novel methodsfor incorporation of production centers into nucleic acid libraries thatmay be used in array analysis. These production centers may provideamplification of sequences that are identical or complementary tosequences in the original diverse nucleic acid analytes. The productsderived from these production centers may be labeled themselves or UDTsmay be incorporated for detection purposes. Nucleic acids that may be ofuse in the present invention can comprise or be derived from DNA or RNA.The original population of nucleic acids may comprise but not be limitedto genomic DNA, unspliced RNA, mRNA, rRNA and snRNA.

This invention provides a composition of matter that comprises a libraryof analytes, the analytes being hybridized to an array of nucleic acids,the nucleic acids being fixed or immobilized to a solid support, whereinthe analytes comprise an inherent universal detection target (UDT), anda universal detection element (UDE) attached to the UDT, wherein the UDEgenerates a signal indicating the presence or quantity of the analytes,or the attachment of UDE to UDT. The library of analytes can be derivedfrom a biological source selected from the group consisting of organs,tissues and cells, or they may be from non-natural sources as discussedin the definitions section above. Biological analytes can be selectedfrom the group consisting of genomic DNA, episomal DNA, unspliced RNA,mRNA, rRNA, snRNA and a combination of any of the foregoing. The nucleicacid array can be selected from the group consisting of DNA, RNA andanalogs thereof, an example of the latter being PNA. Such nucleic acidsor analogs can be modified on any one of the sugar, phosphate or basemoieties. The solid support can take a number of different forms,including being porous or non-porous. A porous solid support can beselected from the group consisting of polyacrylamide and agarose. Anon-porous solid support may comprise glass or plastic. The solidsupport can also be transparent, translucent, opaque or reflective.

Nucleic acids can be directly or indirectly fixed or immobilized to thesolid support. In terms of indirect attachment, the nucleic acids can beindirectly fixed or immobilized to the solid support by means of achemical linker or linkage arm.

As discussed elsewhere in this disclosure, the inherent UDT can selectedfrom the group consisting of 3′ polyA segments, 5′ caps, secondarystructures, consensus sequences and a combination of any of theforegoing. The consensus sequences can be selected from the groupconsisting of signal sequences for polyA addition, splicing elements,multicopy repeats and a combination of any of the foregoing. As alsodiscussed elsewhere in this disclosure, the UDEs can be selected fromthe group consisting of nucleic acids, nucleic acid analogs,polypeptides, polysaccharides, synthetic polymers and a combination ofany of the foregoing. As mentioned previously, such analogs can take theform of PNA. The UDE generates a signal directly or indirectly. Directsignal generation can take any number of forms and can be selected fromthe group consisting of a fluorescent compound, a phosphorescentcompound, a chemiluminescent compound, a chelating compound, an electrondense compound, a magnetic compound, an intercalating compound, anenergy transfer compound and a combination of any of the foregoing.Where indirect signal generation is desired, such can take a number ofdifferent forms and in this regard can be selected from the groupconsisting of an antibody, an antigen, a hapten, a receptor, a hormone,a ligand, an enzyme and a combination of any of the foregoing. Amongsuitable enzymes which can be indirectly detected, these would includeenzymes which catalyze any reaction selected from the group consistingof a fluorogenic reaction, a chromogenic reaction and a chemiluminescentreaction.

This invention also provides a composition of matter that comprises alibrary of analytes, such analytes being hybridized to an array ofnucleic acids, and such nucleic acids being fixed or immobilized to asolid support, wherein the analytes comprise a non-inherent universaldetection target (UDT) and a universal detection element (UDE)hybridized to the UDT, and wherein the UDE generates a signal directlyor indirectly to detect the presence or quantity of such analytes. Thenature of the analyte, the nucleic acid array, modifications, solidsupport are as described in the preceding paragraphs above. Thenon-inherent universal detection targets (UDTs) can comprisehomopolymeric sequences or heteropolymeric sequences. The universaldetection elements (UDEs) can be selected from the group consisting ofnucleic acids, nucleic acid analogs and modified forms thereof. The UDEsgenerate a signal directly or indirectly, such direct and indirectsignal generation also being discussed in the paragraphs just above.

The present invention further provides a composition of matter thatcomprises a library of analytes, such analytes being hybridized to anarray of nucleic acids, and such nucleic acids being fixed orimmobilized to a solid support, wherein the hybridization between theanalytes and the nucleic acids generate a domain for complex formation,and the composition further comprises a signaling entity complexed tothe domain. Statements and features regarding the nature of the libraryof analytes, the nucleic acid array, the solid support and fixation orimmobilization thereto, and direct/indirect signal generation are asdiscussed hereinabove, particularly the last several paragraphs.Notably, the domain for complex formation can be selected from the groupconsisting of DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNAhybrids and RNA-PNA hybrids. The signaling entity that is complexed tothe domain can be selected from the group consisting of proteins andintercalators. Such proteins can comprise nucleic acid binding proteinswhich bind preferentially to double-stranded nucleic acid, the lattercomprising antibodies, for example. These antibodies are specific fornucleic acid hybrids and are selected from the group consisting ofDNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids andRNA-PNA hybrids. In accordance with the present invention, usefulintercalators can be selected from the group consisting of ethidiumbromide, diethidium bromide, acridine orange and SYBR Green. Whenemployed in accordance with the present invention, the proteins generatea signal directly or indirectly. Such forms and manner of direct andindirect signal generation are as described elsewhere in thisdisclosure, particularly in several paragraphs above.

Related to the above described compositions are unique and usefulprocesses. The present invention thus provides a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of: a) providing: (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified, whereineach of the nucleic acids of interest comprise at least one inherentuniversal detection target (UDT); and (iii) universal detection elements(UDE) which generates a signal directly or indirectly; b) hybridizingthe library (ii) with the array of nucleic acids (i) to form hybrids ifthe nucleic acids of interest are present; c) contacting the UDEs withthe UDTs to form a complex bound to the array; d) detecting orquantifying the more than one nucleic acid of interest by detecting ormeasuring the amount of signal generated from UDEs bound to the array.Many of these elements have been described previously in thisdisclosure, but at the risk of some redundancy, elaboration is now made.For example, the nucleic acid array can be selected from the groupconsisting of DNA, RNA and analogs thereof, the latter comprising PNA.Modifications to these nucleic acids and analogs can be usefully carriedout to any one of the sugar, phosphate or base moieties. The solidsupport can be porous, e.g., polyacrylamide and agarose, or non-porous,e.g., glass or plastic. The solid support can also be transparent,translucent, opaque or reflective.

Nucleic acids are directly or indirectly fixed or immobilized to thesolid support. Indirect fixation or immobilization to the solid supportcan be carried out by means of a chemical linker or linkage arm. Asdiscussed elsewhere herein, the library of analytes can be derived froma biological source selected from the group consisting of organs,tissues and cells, or they may be from non-natural or more synthetic orman-made sources. Among biological analytes are those selected from thegroup consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA,rRNA, snRNA and a combination of any of the foregoing.

The inherent UDT used in the above process can be selected from thegroup consisting of 3′ polyA segments, 5′ caps, secondary structures,consensus sequences, and a combination of any of the foregoing. Suchconsensus sequences can be selected from the group consisting of signalsequences for polyA addition, splicing elements, multicopy repeats, anda combination of any of the foregoing. UDEs can be selected from thegroup consisting of nucleic acids, nucleic acid analogs, e.g., PNA,polypeptides, polysaccharides, synthetic polymers and a combination ofany of the foregoing. UDEs generate a signal directly or indirectly.Direct signal generation can be various and may be selected from thegroup consisting of a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chelating compound, an electron densecompound, a magnetic compound, an intercalating compound, an energytransfer compound and a combination of any of the foregoing. Indirectsignal generation can also be various and may be selected from the groupmembers consisting of an antibody, an antigen, a hapten, a receptor, ahormone, a ligand, an enzyme and a combination of any of the foregoing.When desired and employed in the process at hand, such an enzymecatalyzes a reaction selected from the group consisting of a fluorogenicreaction, a chromogenic reaction and a chemiluminescent reaction. Thoseskilled in the art will readily appreciate that the above-describedprocess can further comprise one or more washing steps.

This invention provides another such process for detecting orquantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing: (i) an array of fixed orimmobilized nucleic acids complementary to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified, whereineach of the nucleic acids of interest comprise at least one inherentuniversal detection target (UDT); and (iii) universal detection elements(UDE) which generates a signal directly or indirectly; b) contacting theUDEs with the UDTs in the library of nucleic acid analytes to form oneor more complexes; c) hybridizing the library of nucleic acid analyteswith the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present; d) detecting or quantifying the more thanone nucleic acid of interest by detecting or measuring the amount ofsignal generated from UDEs bound to the array. The nature and form ofthe nucleic acid array, modifications, solid support, direct/indirectfixation or immobilization, library of analytes, inherent UDT, UDE,direct/indirect signal generation, and the like, are as describedelsewhere in this disclosure, including more particularly the lastseveral paragraphs above. Furthermore, this process can comprise one ormore conventional washing steps.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified, wherein each of the nucleic acids of interest comprise atleast one non-inherent universal detection target (UDT), wherein thenon-inherent UDT is attached to the nucleic acid analytes; and (iii)universal detection elements (UDE) which generate a signal directly orindirectly; b) hybridizing the library (ii) with the array of nucleicacids (i) to form hybrids if the nucleic acids of interest are present;c) contacting the UDEs with the UDTs to form a complex bound to thearray; d) detecting or quantifying the more than one nucleic acid ofinterest by detecting or measuring the amount of signal generated fromUDEs bound to the array. As described variously in this disclosure, thenature and form of the nucleic acid array, modifications to nucleic acidand nucleic acid analogs, the solid support, direct and indirectfixation/immobilization to the solid support, the library of analytes,direct and indirect signal generation, and the like, are as describedelsewhere in this disclosure. Of particular mention are the non-inherentuniversal detection targets (UDTs) which can comprise homopolymericsequences and heteropolymeric sequences. Also of particular mention arethe universal detection elements (UDEs) which can be selected from thegroup consisting of nucleic acids, nucleic acid analogs, e.g., PNA, andmodified forms thereof. One or more washing steps can be included inthis last process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified, wherein each of such nucleic acids of interest comprise atleast one non-inherent universal detection target (UDT), wherein thenon-inherent UDTs are attached to the nucleic acid analytes; and (iii)universal detection elements (UDE) which generate a signal directly orindirectly; b) contacting the UDEs with the UDTs in the library ofnucleic acid analytes to form one or more complexes; c) hybridizing thelibrary (ii) with the array of nucleic acids (i) to form hybrids if suchnucleic acids of interest are present; d) detecting or quantifying themore than one nucleic acid of interest by detecting or measuring theamount of signal generated from UDEs bound to the array. Descriptionsfor the nucleic acid array, modifications, solid support,direct/indirect fixation or immobilization to the solid support, thelibrary of analytes, the non-inherent universal detection targets(UDTs), the universal detection elements (UDEs), direct/indirect signalgeneration, inclusion of washing steps, and the like, are foundelsewhere in this disclosure and are equally applicable to this lastdescribed process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) means for attaching one or more universal detectiontargets (UDT) to a nucleic acid; (iv) universal detection elements (UDE)which generates a signal directly or indirectly; b) attaching such UDTs(iii) to the library of nucleic acid analytes (ii); c) hybridizing thelibrary (ii) with the array of nucleic acids (i) to form hybrids if suchnucleic acids of interest are present; d) contacting the UDEs with theUDTs to form a complex bound to the array; e) detecting or quantifyingthe more than one nucleic acid of interest by detecting or measuring theamount of signal generated from UDEs bound to the array. Many of theseelements have been described already. These include the nucleic acidarray, nucleic acid analogs, sugar, phosphate and base modifications,the solid support, direct/indirect fixation and immobilization to thesolid support, the library of analytes, the universal detectionelements, direct/indirect signal generation, inclusion of additionalwashing steps, and the like, have been described elsewhere above andbelow and are equally applicable to this last-mentioned process. Ofspecial mention are attaching means which add homopolymeric sequencesthrough various enzymes, e.g., poly A polymerase and terminaltransferase. Other attaching means can be used for adding homopolymericor heteropolymeric sequences, and these include enzymatic means andenzymes selected from DNA ligase and RNA ligase.

Still another process for detecting or quantifying more than one nucleicacid of interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) means for attaching one or more universal detectiontargets (UDT) to a nucleic acid; (iv) universal detection elements (UDE)which generate a signal directly or indirectly; b) attaching the UDTs(iii) to the library of nucleic acid analytes (ii); c) contacting theUDEs with the UDTs in the library of nucleic acid analytes to form oneor more complexes; d) hybridizing the library (ii) with the array ofnucleic acids (i) to form hybrids if such nucleic acids of interest arepresent; e) detecting or quantifying the more than one nucleic acid ofinterest by detecting or measuring the amount of signal generated fromUDEs bound to the array. As might be expected, the elements recited inthis process have been described elsewhere in this disclosure and areequally applicable to this last described process. These previouslydescribed elements include the nucleic acid array, modifications, thesolid support, direct/indirect fixation or immobilization to the solidsupport, the library of analytes, attaching means, UDE, direct/indirectsignal generation and the inclusion of washing steps.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; and (iii) universal detection elements (UDEs) which bind toa domain formed by nucleic acid hybrids for complex formation andgenerate a signal directly or indirectly; b) hybridizing the library(ii) with the array of nucleic acids (i) to form hybrids if such nucleicacids of interest are present, wherein any formed hybrids generate adomain for complex formation; c) contacting the UDEs with any hybrids toform a complex bound to the array; d) detecting or quantifying the morethan one nucleic acid of interest by detecting or measuring the amountof signal generated from UDEs bound to the array. Descriptions for thenucleic acid array, nucleic acid analogs, e.g., PNA, modifications(sugar, base and phosphate moieties), the solid support,fixation/immobilization, the library of analytes, the domain for complexformation, direct/indirect signal generation from signaling proteins,washing steps, and the like, have already been given above and areequally applicable to this last mentioned process. Of special note isthis process wherein the signaling entity is complexed to the domain forcomplex formation, such signaling entity being selected from proteinsand intercalators. Such proteins can include nucleic acid bindingproteins which bind preferentially to double-stranded nucleic acids,e.g., antibodies, particularly such antibodies which are specific fornucleic acid hybrids, e.g., DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNAhybrids, DNA-PNA hybrids and RNA-PNA hybrids. Intercalators have alsobeen previously described in this disclosure and can be selected fromethidium bromide, diethidium bromide, acridine orange and SYBR Green.

Other compositions of matter are provided by this invention. One suchcomposition comprises a library of first nucleic acid analyte copies,such first nucleic acid copies being hybridized to an array of nucleicacids, those nucleic acids being fixed or immobilized to a solidsupport, wherein such first nucleic acid copies comprise an inherentuniversal detection target (UDT) and a universal detection element (UDE)attached to the UDT, wherein the UDE generates a signal directly orindirectly to detect the presence or quantity of any analytes. Thelibrary of analytes, e.g., biological sources, and examples of suchanalytes, e.g., genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA,snRNA and a combination of any of the foregoing, has been describedabove. Equally so, the nucleic acid array has been already described,including, for example, DNA, RNA and analogs thereof, e.g., PNA.Modifications to the nucleic acids and analogs (sugar, phosphate, base),features of the solid support (porous/non-porous, transparent,translucent, opaque, reflective), fixation/immobilization to the solidsupport, the inherent UDT, the UDE, direct/indirect signal generationfrom UDEs have been described above and apply equally to this lastcomposition.

Another composition of matter comprises a library of first nucleic acidanalyte copies, such first nucleic acid copies being hybridized to anarray of nucleic acids, the nucleic acids being fixed or immobilized toa solid support, wherein such first nucleic acid copies comprise one ormore non-inherent universal detection targets (UDTs) and one or moreuniversal detection elements (UDEs) attached to the UDTs, wherein theUDEs generate a signal directly or indirectly to detect the presence orquantity of any analytes, and wherein the UDTs are either: (i) at the 5′ends of the first nucleic acid copies and not adjacent to an oligoTsegment or sequence, or (ii) at the 3′ ends of the first nucleic acidcopies, or (iii) both (i) and (ii). The library of analytes, nucleicacid array, nucleic acid modifications, solid support,fixation/immobilization to the solid support, non-inherent UDTs, e.g.,heteropolymeric sequences, UDEs (e.g., nucleic acids, nucleic acidanalogs, polypeptides, polysaccharides, synthetic polymers, etc),direct/indirect signal generation from UDEs have already been describedabove and are applicable to this last described composition.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto the nucleic acids of interest; (ii) a library of nucleic acidanalytes which may contain the nucleic acids of interest sought to bedetected or quantified, wherein each of such nucleic acids of interestcomprise at least one inherent universal detection target (UDT); (iii)universal detection elements (UDE) which generate a signal directly orindirectly; and (iv) polymerizing means for synthesizing nucleic acidcopies of the nucleic acids of analytes; b) synthesizing one or morefirst nucleic acid copies which are complementary to all or part of thenucleic acid analytes and synthesizing sequences which are complementaryto all or part of the UDT to form a complementary UDT; c) hybridizingsuch first nucleic acid copies with the array of nucleic acids (i) toform hybrids if such nucleic acids of interest are present; d)contacting the UDEs with the complementary UDTs of the first nucleicacid copies to form a complex bound to the array; e) detecting orquantifying the more than one nucleic acid of interest by detecting ormeasuring the amount of signal generated from UDEs bound to the array.Statements and descriptions for the nucleic acid array, modifications,solid support, fixation/immobilization, the library of analytes,inherent UDTs, e.g., consensus sequences, UDEs, direct/indirect signalgeneration from UDEs, have been given above and are equally applicableto this last process. Of special mention are the recited polymerizingmeans which can be selected from E. coli DNA Pol 1, Klenow fragment ofE. coli DNA Pol 1, Bst DNA polymerase, Bca DNA polymerase, Taq DNApolymerase, Tth DNA Polymerase, T4, DNA polymerase, ALV reversetranscriptase, MuLV reverse transcriptase, RSV reverse transcriptase,HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscriptand Omniscript.

Another embodiment provided by this invention is a process for detectingor quantifying more than one nucleic acid of interest in a librarycomprising the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical in part or whole to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified, wherein each of such nucleic acids of interest comprise atleast one inherent universal detection target (UDT); (iii) universaldetection elements (UDE) which generate a signal directly or indirectly;and (iv) polymerizing means for synthesizing nucleic acid copies of suchnucleic acid analytes; b) synthesizing one or more first nucleic acidcopies of such nucleic acid analytes; c) contacting the UDEs with theUDTs in the first nucleic acid copies to form one or more complexes; d)hybridizing such first nucleic acid copies with the array of nucleicacids (i) to form hybrids if such nucleic acids of interest are present;and e) detecting or quantifying the more than one nucleic acid ofinterest by detecting or measuring the amount of signal generated fromUDEs bound to the array. The nucleic acid array, nucleic acidmodifications, the solid support, fixation/immobilization (direct andindirect), the library of analytes, inherent UDTs, UDEs, signalgeneration from UDEs (direct/indirect), polymerizing means, have beendescribed above. Such descriptions are equally applicable to this lastprocess.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto the nucleic acids of interest; (ii) a library of nucleic acidanalytes which may contain the nucleic acids of interest sought to bedetected or quantified; (iii) means for attaching one or morenon-inherent universal detection targets (UDT) to a nucleic acid; (iv)universal detection elements (UDE) which generate a signal directly orindirectly; and (v) polymerizing means for synthesizing nucleic acidcopies of the nucleic acid analytes; b) attaching the non-inherent UDtsto either the 3′ ends of the nucleic acid analytes, the 5′ ends of thefirst nucleic acid analytes, or both the 3′ ends and the 5′ ends of thenucleic acid analytes; c) synthesizing one or more first nucleic acidcopies of the nucleic acid analytes; d) hybridizing the first nucleicacid copies with the array of nucleic acids (i) to form hybrids if suchnucleic acids of interest are present; e) contacting the UDEs with theUDTs of the first nucleic acid copies to form a complex bound to thearray; and f) detecting or quantifying the more than one nucleic acid ofinterest by detecting or measuring the amount of signal generated fromUDEs bound to the array. See many of the preceding paragraphs fordescriptions and characteristics of the nucleic acid array,modifications, the solid support, fixation/immobilization, the libraryof analytes, attaching means, UDEs, direct/indirect signal generationfrom UDEs, polymerizing means, and the like.

Yet another process for detecting or quantifying more than one nucleicacid of interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto the nucleic acids of interest; (ii) a library of nucleic acidanalytes which may contain the nucleic acids of interest sought to bedetected or quantified; (iii) means for attaching one or morenon-inherent universal detection targets (UDT) to a nucleic acid; (iv)universal detection elements (UDE) which generate a signal directly orindirectly; and (v) polymerizing means for synthesizing nucleic acidcopies of the nucleic acid analytes; b) attaching such non-inherent UDTsto either the 3′ ends of the nucleic acid analytes, the 5′ ends of thefirst nucleic acid analytes, or both the 3′ ends and the 5′ ends of thenucleic acid analytes; c) synthesizing one or more first nucleic acidcopies of the nucleic acid analytes; d) contacting the UDEs with theUDTs of the first nucleic acid copies to form complexes; e) hybridizingthe first nucleic acid copies with the array of nucleic acids (i) toform hybrids if any nucleic acids of interest are present; f) detectingor quantifying the more than one nucleic acid of interest by detectingor measuring the amount of signal generated from UDEs bound to thearray. The nucleic acid array, modifications, the solid support,direct/indirect fixation/immobilization, the library of analytes,attachment means, UDEs, signal generation from UDEs, direct/indirectsignal generation, polymerizing means, and the like, have already beendescribed. Such descriptions are equally applicable to thislast-described process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto such nucleic acids of interest; (ii) a library of nucleic acidanalytes which may contain the nucleic acids of interest sought to bedetected or quantified; (iii) means for attaching one or morenon-inherent universal detection targets (UDT) to a nucleic acid; (iv)universal detection elements (UDE) which generate a signal directly orindirectly; and (v) polymerizing means for synthesizing nucleic acidcopies of the nucleic acid analytes; b) synthesizing one or more firstnucleic acid copies of the nucleic acid analytes; c) attaching thenon-inherent UDTs to either the 3′ ends of the first nucleic acidcopies, the 5′ ends of the first nucleic acid copies, or both the 3′ends and the 5′ ends of the first nucleic acid copies; d) hybridizingthe first nucleic acid copies with the array of nucleic acids (i) toform hybrids if any nucleic acids of interest are present; e) contactingthe UDEs with the UDTs of the first nucleic acid copies to form acomplex bound to the array; and f) detecting or quantifying the morethan one nucleic acid of interest by detecting or measuring the amountof signal generated from UDEs bound to the array. Descriptions for theabove-recited elements have been given above and are equally applicableto this last process.

Still another process provided by this invention is for detecting orquantifying more than one nucleic acid of interest in a librarycomprises the steps of a) providing (i) an array of fixed or immobilizednucleic acids identical in part or whole to the nucleic acids ofinterest; (ii) a library of nucleic acid analytes which may contain thenucleic acids of interest sought to be detected or quantified; (iii)means for attaching one or more non-inherent universal detection targets(UDT) to a nucleic acid; (iv) universal detection elements (UDE) whichgenerate a signal directly or indirectly; and (v) polymerizing means forsynthesizing nucleic acid copies of the nucleic acid analytes; b)synthesizing one or more first nucleic acid copies of the nucleic acidanalytes; c) attaching the non-inherent UDTs to either the 3′ ends ofthe first nucleic acid copies, the 5′ ends of the first nucleic acidcopies, or both the 3′ ends and the 5′ ends of the first nucleic acidcopies; d) contacting the UDEs with the UDTs of the first nucleic acidcopies to form a complex; e) hybridizing the first nucleic acid copieswith the array of nucleic acids (i) to form hybrids if any nucleic acidsof interest are present; and f) detecting or quantifying the more thanone nucleic acid of interest by detecting or measuring the amount ofsignal generated from UDEs bound to the array. These elements andsubelements have been described elsewhere in this disclosure. Suchdescriptions apply to this last process.

Yet another process for detecting or quantifying more than one nucleicacid of interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids complementary to the nucleicacids of interest; (ii) a library of nucleic acid analytes which maycontain the nucleic acids of interest sought to be detected orquantified; (iii) universal detection elements (UDEs) which bind to adomain for complex formation formed by nucleic acid hybrids and generatea signal directly or indirectly; and (iv) polymerizing means forsynthesizing nucleic acid copies of the nucleic acid analytes; b)synthesizing one or more nucleic acid copies of the nucleic acidanalytes; c) hybridizing the first nucleic acid copies with the array ofnucleic acids (i) to form hybrids if any nucleic acids of interest arepresent, wherein any formed hybrids generate a domain for complexformation; d) contacting the UDEs with the hybrids to form a complexbound to the array; and e) detecting or quantifying the more than onenucleic acid of interest by detecting or measuring the amount of signalgenerated from UDEs bound to the array. The above-recited elements andsubelements and variations thereof are described elsewhere in thisdisclosure and are equally applicable to this just-mentioned process.

One aspect of the present invention discloses methods that eliminate thenecessity for enzymatic incorporation of labeled nucleotides by an enduser. In this particular aspect, common or conserved features present ina diverse population of nucleic acid analytes are used to assay theextent of hybridization of the analytes to discrete target elements inan array format. These common or conserved features are UniversalDetection Targets (UDTs) which can provide signal generation by bindingof Universal Detection Elements (UDEs).

Examples of UDTs that may be inherently present in a population ofdiverse nucleic acid analytes can comprise but not be limited to 3′ polyA segments, 5′ caps, secondary structures and consensus sequences.Examples of consensus sites that might find use in the present inventioncan comprise but not be limited to signal sites for poly A addition,splicing elements and multicopy repeats such as Alu sequences.

UDEs may be directly or indirectly labeled. Examples of directly labelscan comprise but not be limited to any members of a group consisting ofa fluorescent compound, a phosphorescent compound, a chemiluminescentcompound, a chelating compound, an electron dense compound, a magneticcompound, an intercalating compound, an energy transfer compound and acombination of any of the foregoing.

Examples of indirect labels can comprise but not be limited to anymembers of a group consisting of an antibody, an antigen, a hapten, areceptor, a hormone, a ligand, an enzyme and a combination of any of theforegoing. Among such enzymes are any enzymes which catalyze reactionsselected from the group consisting of a fluorogenic reaction, achromogenic reaction and a chemiluminescent reaction.

RNA and DNA polymerases sometimes have difficulty in accepting labelednucleotides as substrates for polymerization. In prior art, thisshortcoming can result in the production of a labeled library thatconsists of short strands with few signal generating entities.Limitations caused by such inefficient incorporation can be partiallycompensated for by increasing the amount of labeled precursors in thereaction mixtures. However, this method achieves only a moderateimprovement and entails a higher cost and waste of labeled reagents. Incontrast, this particular aspect of the present invention disclosesmeans by which diverse nucleic acids in a library can be hybridized inan array format in their native form without the need of anymanipulations or modifications and then be detected by the presence ofUDTs bound to the array.

An illustrative depiction of this process is given in FIG. 1. Althoughthere are multiple unique species of mRNA that can make up a diversepopulation of nucleic acids in a sample, the common elements that areshared by these nucleic acids can be used as UDTs. Hybridization of themRNA to an array permits the localization of individual species todiscrete locations on the array. The determination of the amount ofsample that is bound to each locus of an array is then carried out bydetection of the amount of UDT present at each locus by binding of theappropriate UDE. Thus, in FIG. 1, locus 1 and 3 would be capable ofgenerating an amount of signal that would be proportionate to the amountof mRNA bound to each of those sites. On the other hand there wouldlittle or no signal generation from locus 2 since there was little or nomRNA bound to that site. A single labeled species of mostly orcompletely poly T or U could be used as a UDE to quantify the amount ofpoly A tails of the various species of eucaryotic mRNA in FIG. 1. Inthis way, a single universal species of labeled material is synthesizedfor use as a UDE thereby providing an inexpensive and efficient means ofindirectly labeling the RNA molecules being quantified.

A nucleic acid UDE can be prepared either chemically or enzymatically.For example, oligonucleotide synthesizers are commercially availablethat can produce a UDE consisting of labeled poly T/U sequences fordetection of the poly A UDT described above. Both the amount andplacement of labeled moieties can be tightly controlled by this method.Also, since this is a homopolymeric product, probes that are shorter byone or more bases will still be effective such that the net yield ofusable product will be higher than one that requires a discrete specificsequence. On the other hand, methods of synthesizing such sequencesenzymatically are also well known to those versed in the art. Commonly,a tetramer of dT is used as a primer for addition of poly T or poly U byterminal transferase. Each base can be modified to be capable of signalgeneration or a mixture of labeled and unlabeled bases can be used.Although A Poly A UDT has been described in the example above, whendifferent sequences are used as UDTs, the synthesis of the corespondingUDEs can be carried out by the same chemical and enzymatic methodologiesdescribed above. It is also contemplated that analogues of DNA can alsobe used to synthesize the UDEs. For instance, instead of using DNA,labeled RNA or PNA (peptide nucleic acids) may also be used.

Detection and quantification of the amount of UDTs bound to particularloci can also be carried out by the use of an antibody acting as a UDE.Examples of antibody specificities that are useful for UDEs can comprisebut not be limited to recognition of the cap element at the 5′ end ofmature mRNAs or the homopolymeric poly A sequence. Furthermore,hybridization between nucleic acids is an event that in and of itself iscapable of generating a UDT that can be recognized by antibody UDEs. Forexample, when a library of diverse RNA species are bound to an array,the RNA, DNA or PNA target elements in the array will generate RNA/RNA,RNA/DNA or RNA/PNA hybrids at each of the loci that has homology withthe particular RNA species being quantified. Although each of the siteshas a discrete sequence, universal detection and quantification can becarried out by antibodies that recognize the change in physicalstructure produced by such hybridization events. Alternatively, thehybridization between a UDE and the complementary UDT of a nucleic acidbound to the target elements of the array can be detected by anappropriate antibody. The antibodies that are specific for the UDEsdescribed above can be labeled themselves or secondary labeledantibodies can be used to enhance the signal.

If only a single library of mRNA is being analyzed, binding of a UDE toa UDT may take place before or after hybridization of the RNA to anarray of detection probes. The particular order of events will dependupon the nature and stability of the binding partners. When analytesfrom two libraries are intended to be compared simultaneously, bindingof each UDE to a binding partner is preferably carried out prior tohybridization of the RNA to an array of target elements such that eachlibrary is differentially labeled. Although comparisons are typicallycarried out between two libraries, any number of comparisons can be madesimultaneously as long as each library is capable of generating a signalthat can be distinguished from the other libraries. On the other hand,rather than simultaneous hybridization and detection, the arrays can beused in a parallel or sequential fashion. In this format, hybridizationand detection is carried out separately for each library and theanalysis of the results is compared afterwards relative to normalizedcontrols of steady state genes.

In another aspect of the present invention, UDTs or UDEs areartificially incorporated into the diverse nucleic acids of the library.Enzymes that find particular use with RNA analytes may comprise but notbe limited to Poly A polymerase which specifically adds Adenineribonucleotides to the 3′ end of RNA and RNA ligase which can add anoligonucleotide or polynucleotide to either the 5′ or 3′ end of an RNAanalyte. By these means, either homopolymeric or unique sequences can beadded to serve as UDTs or UDEs. Enzymes that find particular use withDNA analytes may comprise but not be limited to Terminal Transferase foraddition to 3′ ends and DNA ligase for addition to either 3′ or 5′ ends.The sequences that are introduced into the nucleic acid analytes can belabeled during synthesis or addition of a UDE or conversely unlabeledUDTs can be synthesized or added that are detected later bycorresponding labeled UDEs. This aspect enjoys special utility whenunspliced RNA, snRNA, or rRNA are used as analytes since they may belacking inherent elements that are present in mRNA that have previouslycited as being useful as UDTs. This aspect of the present invention willalso find use with procaryotic mRNA since the poly A additions, 5′ capsand splicing elements which have been previously cited as potential UDTsof mRNA are intrinsically lacking in procaryotes.

This particular aspect of the present invention may also be used inconjunction with fragmentation processes. For instance, mRNA moleculesfrom eucaryotic organisms can be very large even after processing eventshave taken place. This size factor can hinder hybridization or allowscissions between the segment used for binding to a target element inthe array and the UDT that is being used for signal generation.Additionally, a fragmentation step may also reduce the amount ofsecondary structure present in RNA. Therefore, in this aspect of thepresent invention, RNA can be fragmented into smaller sized pieceseither by physical or enzymatic followed by addition of sequences thatcan act as UDTs or UDEs. Examples of physical means for fragmentation ofnucleic acids can include but not be limited to shearing or alkalitreatment. Examples of enzymatic means can include but not be limited toa partial nuclease or RNase digestion.

In addition, DNA from most sources will also be extremely large in itsnative form. DNA analytes may also be fragmented by suitable physical orenzymatic means. A particularly useful enzymatic means would be the useof restriction enzymes where the nature of the recognition sequence forthe restriction enzyme will determine the average size of the fragments.Also, although most restriction enzymes require double-stranded DNA astemplates, some enzymes such as Hha I, Hin P1 I and Mnl I cleavesingle-stranded DNA efficiently (2000-2001 catalog, New England BioLabs,Beverly, Mass., p214). By this fragmentation method a single analytemolecule is converted into multiple subfragments that can each havetheir own artificially introduced UDT or UDE. An exemplary illustrationof this particular aspect of the present invention is included in FIG.2.

In another aspect of the present invention, the diverse nucleic acids ina library are used as templates for synthesis of complementary nucleicacid copies instead of using the analytes directly for array analysis.The analyte templates may have intrinsic UDTs present or they may haveUDTs artificially incorporated by the means cited earlier. On the otherhand, the UDTs do not have to be present in the analyte templates andincorporation of artificial UDTs can take place either during or aftersynthesis of nucleic acid copies. Examples of enzymes that may be usedfor making copies of DNA templates can comprise but not be limited toDNA polymerases for synthesis of DNA copies, and RNA polymerases for thesynthesis of RNA copies. Examples of DNA polymerases that may have usein the present invention for synthesis of DNA copies from DNA templatescan include but not be limited to E. coli DNA Pol I, the Klenow fragmentof E. coli DNA Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNApolymerase, Tth DNA polymerase, T4 DNA polymerase, T7 DNA polymerase,ALV Reverse Transcriptase, RSV Reverse Transcriptase, HIV-1 ReverseTranscriptase, HIV-2 Reverse Transcriptase, Sensiscript, Omniscript andvarious mutated or otherwise altered forms of the foregoing. Examples ofRNA polymerases that may have use in the present invention for synthesisof RNA copies from DNA templates can include but not be limited tobacteriophage T3 RNA polymerase, bacteriophage T7 RNA polymerase andbacteriophage SP6 RNA polymerase. Examples of enzymes that may have usein the present invention for making DNA copies of RNA templates cancomprise but not be limited to ALV Reverse Transcriptase, RSV ReverseTranscriptase, HIV-1 Reverse Transcriptase, HIV-2 Reverse Transcriptase,Sensiscript, Omniscript, Bst DNA polymerase, Bca DNA polymerase, Tth DNApolymerase and various mutated or otherwise altered forms of theforegoing.

Examples of enzymes that may have use in the present invention formaking RNA copies of RNA templates can comprise but not be RNA dependentRNA polymerases (Koonin, 1991 J. Gen Virol. 72; 2197-2206, incorporatedherein by reference).

Efficient synthesis of complementary copies of analyte templates requirethe presence of a promoter for efficient synthesis by DNA dependent RNApolymerases while the other cited exemplary enzymes require primers.Incorporation of a UDT into a DNA analyte that will be transcribed by aDNA dependent RNA polymerase can comprise but not be limited to ligationof a UDT sequence and a promoter sequence by the action of DNA ligase.This process is depicted below:DNA analyte+UDT-Promoter=DNA Analyte-UDT-Promoter

Transcription of this construct would then be capable of production ofRNA with the structure: 3′ analyte-UDT 5′:

One means of carrying out this particular aspect of the presentinvention is digestion of a library of diverse double-stranded DNAanalytes by a restriction enzyme followed by ligation of adouble-stranded DNA segment comprising an RNA promoter sequence.Subsequent transcription of the transcription units can synthesizeeither labeled or unlabeled transcripts. The unlabeled transcripts canbe detected by the presence of either inherent or synthetically addedUDTs.

When primers are used for synthesis of complementary copies of analytetemplates, the primers can comprise random sequences or selectedsequences for binding to the analyte templates. Random primers that havecommonly been used for priming events have ranged from hexamers tododecamers. Selected sequences that are useful as primers can becomplementary to inherent sequences or to non-inherent sequences thathave been introduced into the analyte templates. Examples of inherentsequences can include but not be limited to consensus sequences orhomopolymeric sequences. Consensus sequences can be derived fromelements that are retained in a large portion of the population beingstudied. Examples of these could comprise but not be limited to poly Aaddition sites, splicing elements and multicopy repeats such as Alusequences. An example of inherent homopolymeric sequences used forprimer binding can be the poly A tail that is intrinsic to mature mRNAin eucaryotes. Non-inherent homopolymeric or unique sequences that canbe used for primer binding may be introduced into RNA templates by meansthat can include but not be limited to poly A polymerase or RNA ligase.Non-inherent homopolymeric or unique sequences that can be used forprimer binding may be introduced into DNA templates by means that caninclude but not be limited to Terminal Transferase and DNA ligase. Theartificial binding sites can be introduced into intact nucleic acidtemplates or fragmentation processes may be carried out as describedpreviously.

When homopolymeric or conserved sequences are used as primer bindingsites, the library can be subdivided by the use of primers that havebeen synthesized with 1 or more additional discrete bases at the 3′ end.For example, an oligonucleotide primer that has the formula5′-T_(n)dC-3′ would preferentially prime mRNAs whose last base was a Gbefore the poly A tail rather than priming the entire population ofmRNA's with poly A tails. The same principle would also hold true wheneither 5′-T_(n)dG-3′ or 5′-T_(n)dA-3′ primers are used. This wouldprovide three separate sub-populations of copies of the original mRNApopulation that in toto should encompass the entire RNA population withpoly A tails. This population could be further divided by inclusion of a2^(nd) discrete base at the 3′ end of the primers. In this case,oligonucleotides would have either dC, dG, dA or dT as the last base atthe 3′ end and dC, dG or dA in the penultimate position and theremaining portion comprising a poly T segment. This would create thepotential for 12 separate pools from the original population. Furtherprovision of discrete bases at the 3^(rd) nucleotide position from the3′ end would provide a separation into 48 different subpopulations ifdesired and so on.

The use of subpopulations may have utility in providing RNA with lowercomplexity thereby simplifying analysis later on. In addition, the useof discrete bases at the 3′ end would limit the size of poly T tails atthe end of the cDNA copies since significant amounts of priming eventswill only take place at the junction of the poly A addition site. Thismay reduce background hybridization caused by extensive polyT or PolyAtracts. Also it may increase yields of labeled products by decreasingstalling or premature terminations caused by long homopolymeric tracts.On the other hand, the use of a mixture of oligo T primers with discretebases at the 3′ end would be similar to a completely homopolymeric oligoT primer in being able to synthesize a complete representation of theoriginal analyte sequences while at the same retaining the ability toconstrain the size of homopolymeric tails.

In this particular aspect of the present invention, the cDNA moleculessynthesized from the pool of RNA templates also comprise UDTs or UDEs.As described previously, these UDTs can be inherently present or theymay be non-inherent sequences that are artificially incorporated duringsynthesis of cDNA. When an analyte has a nucleic acid sequence that canbe used as a UDT, synthesis of the complementary copy creates a sequencethat can also be used as a UDT. For example, the poly A sequence at the3′ end of eucaryotic mRNA was previously described as a potential UDT.When this mRNA is used as a template by extension of a poly T primerwith or without additional bases, the poly T segment of the cDNA copycan function as a UDT. The destruction or separation of the RNAtemplates from the cDNA would allow the poly T at the 5′ end of the cDNAto act as a UDT by binding of a labeled poly A UDE. UDTs or UDEs canalso be incorporated into cDNA copies by inclusion of nucleic acidsegments that don't participate in primer binding into the 5′ tails ofeither random, homopolymeric, or specific sequence primers. Theparticular sequence of the additional nucleic acid segments used as UDTsare of arbitrary nature since they aren't needed for primer binding. Assuch, the choice of sequence for these UDTs can range in complexity fromhomopolymeric sequences to specific unique sequences. Their nature isalso arbitrary, and either the primer or the UDT can comprise PNA's orother nucleic acid homologues. In addition, they may be other polymericentities besides nucleic acids that provide recognition for UDEs.

Since the nature of the UDT or UDE can be selected by the user, thepresent invention allows simple differentiation between libraries thatare being compared. For instance, one population that is being studiedcan be extended by homopolymeric or random primers and hybridized with aUDE labeled with Cy 3. A second population that is being compared can beextended by homopolymeric or random primers and hybridized with UDEsthat have Cy 5 incorporated into them. The other end of the cDNA is alsoavailable for use with UDEs. For example, after synthesis of cDNA copiesby reverse transcriptase, the 3′ ends can be extended further by thenon-template directed addition of nucleotides by Terminal Transferase.An illustration of this particular aspect of the present invention isincluded in FIG. 3.

Detection of the presence of UDTs or UDEs in the library or libraries ofvarious nucleic acids can be carried out by any of the means that havebeen described previously for UDTs. If only a single library is beinganalyzed, binding of a probe or antibody to a 5′ or 3′ UDT or UDE maytake place before or after hybridization of nucleic acids to thedetection elements of the array. The particular order of events willdepend upon the nature and stability of the binding partners. On theother hand, when each population incorporates a different UDT or UDE,binding of labeled moieties to the UDTs can take place either before orafter hybridization of the copies of the analyte to an array. However,as described previously, the same UDT or UDE can be used for eachpopulation if parallel or sequential hybridizations are carried out.

It is also contemplated that the various aspects of the presentinvention can be used to augment rather than substitute for otherpreviously disclosed methods. For instance, signal can be generated incDNA copies by a labeled primer being extended in the presence oflabeled nucleotides. The signal generated by such a method would be asummation of the signal generated by the original primer and whateverlabeled nucleotides were incorporated during strand extension. Thus, acombination of methodologies would generate a signal that would behigher than the amount that would be achieved by either method alone. Inaddition to a pre-labeled primer, the other methods that are disclosedin the present invention can also be used in various combinations.

There may be situations where amplification of sequences in a sample isadvantageous. Therefore, in another aspect of the present invention,multiple cycles of synthesis can be carried out to generate linearamplification of a library of diverse nucleic acid sequences. In thefirst step of this particular aspect of the present invention, theentire population or a subset of the population of nucleic acidsanalytes is used to synthesize 1^(st) strand nucleic acid copies.Whether the initial analyte is DNA or RNA, in the context of the presentinvention, this product is considered to be a cNA since it represents anucleic acid copy of the analyte. Synthesis of the 1^(st) strand nucleicacid copies can be carried out as described previously by using discreteprimers, random primers, homopolymers, or homopolymers with one or morediscrete bases at their 3′ ends. In this particular embodiment of thepresent invention, priming with homopolymers with one or more discretebases at their 3′ ends may also increase the efficiency of amplificationsince resources such as primers and substrates will be directed onlytowards amplification of a discrete subpopulation derived from the1^(st) cNA synthesis reaction.

For linear amplification, a primer binding site on a nucleic acidanalyte is used multiple times by separation of a 1^(st) cNA copy fromits template followed by reinitiation of a new 1^(st) cNA copy.Separation can be carried out by exposure of the reaction mix to hightemperature. If the enzyme used for nucleic acid synthesis is Taqpolymerase, Tth polymerase or some other heat stable polymerase themultiple reactions can be carried out by thermocycling of the reactionwithout the addition of any other reactions. On the other hand, if highdenaturation temperatures are used in conjunction with enzymes that areheat labile, for instance Bst DNA polymerase, Klenow fragment of Pol Ior MuLV Reverse Transcriptase, irreversible heat inactivation of theenzyme takes place and the enzyme has to be replenished for furtherrounds of cNA synthesis. Alternatively, methods have been disclosed byFuller in U.S. Pat. No. 5,432,065 and by Lakobashvill and Lapidot, 1999(Nucleic Acids Research 27; 1566-1568) for reagents that allow lowtemperature denaturation of nucleic acids for use with PCR, both ofwhich methods are incorporated by reference. Furthermore, Winhoven andRossau have disclosed in PCT Application WO 98/45474 (also incorporatedby reference) that temperature manipulation can be avoided completely byelectrically controlled manipulation of divalent ion levels. Thus bythese methods even thermo-labile enzymes can carry out multiple cyclesof synthesis for linear amplification. Both above-cited patent documentsand the above-cited publication are incorporated herein by reference.

Amplification is a significant aspect of this invention. Severalcompositions and processes are devoted and directed to amplification.For example, provided herein is a composition of matter comprising alibrary of double-stranded nucleic acids substantially incapable of invivo replication and free of non-inherent homopolymeric sequences, thenucleic acids comprising sequences complementary or identical in part orwhole to inherent sequences of a library obtained from a sample, whereinthe double-stranded nucleic acids comprise at least one inherentuniversal detection target (UDT) proximate to one end of the doublestrand and at least one non-inherent production center proximate to theother end of the double strand. The sample from which the inherentsequences of the library are obtained can comprise biological sources,e.g., organs, tissues and cells. As described elsewhere herein, thelibrary of nucleic acids can be derived from genomic DNA, episomal DNA,unspliced RNA, mRNA, rRNA, snRNA and a combination of any of theforegoing. Inherent UDTs can be selected from the group consisting of 3′polyA segments, consensus sequences, or both. As already describedabove, consensus sequences can be selected from the group consisting ofsignal sequences for poly A addition, splicing elements, multicopyrepeats, and a combination of any of the foregoing. Of special mentionis the production center which can be selected from the group consistingof primer binding sites, RNA promoters, or a combination of both. SuchRNA promoters can comprise phage promoters, e.g., T3, T7 and SP6.

Another composition of matter for amplification purposes comprises alibrary of double-stranded nucleic acids substantially incapable of invivo replication, such nucleic acids comprising sequences complementaryor identical in part or whole to inherent sequences of a libraryobtained from a sample, wherein the double-stranded nucleic acidscomprise at least four (4) non-inherent nucleotides proximate to one endof the double strand and a non-inherent production center proximate tothe other end of the double strand. Descriptions for such elements,i.e., the sample, the library of nucleic acids, inherent UDTs,non-inherent nucleotides, non-inherent production centers, e.g., RNApromoters, e.g., phage promoters (T3, T7 and SP6) are given elsewhere inthis disclosure and are equally applicable to this last composition.

Another composition of matter for amplification comprises a library ofdouble-stranded nucleic acids fixed to a solid support, those nucleicacids comprising sequences complementary or identical in part or wholeto inherent sequences of a library obtained from a sample and thenucleic acids further comprising at least one first sequence segment ofnon-inherent nucleotides proximate to one end of the double strand andat least one second sequence segment proximate to the other end of thedouble strand, the second sequence segment comprising at least oneproduction center. Of special mention is the use of beads as the solidsupport, particularly beads and magnetic beads. Other elements, such asthe sample and biological sources, the library of nucleic acids,inherent UDTs, non-inherent production centers, have already beendescribed.

Yet another amplification type composition of matter comprises a libraryof double-stranded nucleic acids attached to a solid support, thenucleic acids comprising sequences complementary or identical in part orwhole to inherent sequences of a library obtained from a sample, whereinthe double-stranded nucleic acids comprise at least one inherentuniversal detection target (UDT) proximate to one end of the doublestrand and at least one non-inherent production center proximate to theother end of the double strand. The elements and subelements (solidsupport, beads, magnetic beads, sample, library of nucleic acids,inherent UDTs, consensus sequences, production centers, RNA promoters,phage promoters, e.g., T3, T7 and SP6, have been described above.

Among useful processes for detecting or quantifying more than onenucleic acid of interest in a library, one such process of the presentinvention comprises the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, the polymerizing meanscomprising a first set of primers and a second set of primers, whereinthe second set of primers comprises at least two segments, the firstsegment at the 3′ end comprising random sequences, and the secondsegment comprising at least one production center; (iv) means forsynthesizing nucleic acid copies under isothermal or isostaticconditions; b) contacting the library of nucleic acid analytes with thefirst set of primers to form more than one first bound entity; c)extending the bound first set of primers by means of template sequencesprovided by the nucleic acid analytes to form first copies of theanalytes; d) contacting the extended first copies with the second set ofprimers to form more than one second bound entity; e) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; f)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; g) hybridizing any nucleic acid copies formed instep f) to the array of nucleic acids provided in step a) (i); and h)detecting or quantifying any of the hybridized copies obtained in stepg). Elements recited in the process just above and their subelementshave already been described in this disclosure. Of special mention isthe first set of primers which are complementary to inherent UDTs.Further mention should be made that the hybridized nucleic acids cancomprise one or more signaling entities attached or incorporatedthereto. As described variously above, signal detection can be carriedout directly or indirectly. Mention is also made that the process canfurther comprise the step of separating the first copies obtained fromstep c) from their templates and repeating step b). Other steps can alsobe included such as the step of separating the extended second set ofprimers obtained from step f) from their templates and repeating stepe). Step g) can also be carried out repeatedly, a feature provided bythis invention and this last-described process. Further, means forsynthesizing nucleic acid copies under isothermal or isostaticconditions is carried out by one or more members selected from the groupconsisting of RNA transcription, strand displacement amplification andsecondary structure amplification. These are all contemplated for use ofthis process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; (iii) polymerizing meansfor synthesizing nucleic acid copies of the nucleic acid analytes, suchpolymerizing means comprising a first set of primers and a second set ofprimers, wherein the first set of primers comprise at least oneproduction center; and (iv) means for synthesizing nucleic acid copiesunder isothermal or isostatic conditions; b) contacting the library ofnucleic acid analytes with the first set of primers to form more thanone first bound entity; c) extending the bound first set of primers bymeans of template sequences provided by the nucleic acid analytes toform first copies of the analytes; d) extending the first copies bymeans of at least four (4) or more non-inherent homopolymericnucleotides; e) contacting the extended first copies with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; g)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph). Of special mention is the use or addition of terminal transferase inor after extending step d) wherein the four or more non-inherenthomopolymeric nucleotides are themselves added. Elements and subelementsof this process are described above. Special mention is made of certainaspects of this process. For example, means for synthesizing nucleicacid copies under isothermal or isostatic conditions can be carried outby one or more members selected from the group consisting of RNAtranscription, strand displacement amplification and secondary structureamplification. Moreover, the step of separating the first copiesobtained from step c) from their templates and repeating step b) can beadded to this process. Moreover, the extended second set of primersobtained from step f) can be separated from their templates and thenstep e) can be repeated as necessary or desired. In fact, step g) can berepeated as often as desired or deemed necessary.

A process for detecting or quantifying more than one nucleic acid ofinterest in a library comprises the steps of a) providing (i) an arrayof fixed or immobilized nucleic acids identical or complementary in partor whole to sequences of the nucleic acids of interest; (ii) a libraryof nucleic acid analytes which may contain the nucleic acids of interestsought to be detected or quantified; (iii) polymerizing means forsynthesizing nucleic acid copies of the nucleic acid analytes, suchpolymerizing means comprising a first set of primers and a second set ofprimers, wherein the first set comprises at least one production center;(iv) a set of oligonucleotides or polynucleotides complementary to atleast one segment or sequence of the second set of primers; and (v)means for ligating the set of oligonucleotides or polynucleotides (iv);b) contacting the library of nucleic acid analytes with the first set ofprimers to form more than one first bound entity; c) extending the boundfirst set of primers by means of template sequences provided by thenucleic acid analytes to form first copies of the analytes; d) ligatingthe set of oligonucleotides or polynucleotides a) (iv) to the 3′ end ofthe first copies formed in step c) to form more than one ligatedproduct; e) contacting the ligated product with the second set ofprimers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph). Aspects of this process, including the nucleic acid array,modifications, solid support, fixation/immobilization, nucleic acidanalytes, UDTs, production centers, signal generation, polymerizingmeans, additional steps and repeating steps, synthesizing means, and soforth, have been described above and apply equally to thislast-mentioned process. Of special mention are the above-recitedligating means which can comprise, for example, T4 DNA ligase.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; (iii) polymerizing meansfor synthesizing nucleic acid copies of the nucleic acid analytes, suchpolymerizing means comprising a first set of primers and a second set ofprimers, wherein the second set comprises at least one productioncenter; (iv) a set of oligonucleotides or polynucleotides complementaryto at least one segment or sequence of the second set of primers; and(v) means for ligating the set of oligonucleotides or polynucleotides(iv); b) contacting the library of nucleic acid analytes with the firstset of primers to form more than one first bound entity; c) extendingthe bound first set of primers by means of template sequences providedby the nucleic acid analytes to form first copies of the analytes; d)ligating the set of oligonucleotides or polynucleotides a) (iv) to the3′ end of the first copies formed in step c) to form more than oneligated product; e) contacting the ligated product with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe ligated products formed in step d) to form more than one complexcomprising the ligated products and the extended second set of primers;g) synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph). Each of the above-recited elements in this process have beendescribed elsewhere in this disclosure. Such descriptions are equallyapplicable to this process. Of special mention is the process whereinthe first set of primers comprise one or more sequences which arecomplementary to inherent UDTs. The hybridized nucleic acid copies canfurther comprise one or more signaling entities attached or incorporatethereto. If so, previously described embodiments for signal generationand detection, e.g., direct and indirect generation and detection, areapplicable to this process. As described previously for other similarprocesses, additional steps can be carried out. For example, the step ofseparating the first copies obtained from step c) from their templatesand then repeating step b) can be carried out. A further step ofseparating the extended second set of primers obtained from step f) fromtheir templates and then repeating step e) can be carried out. Also,step g) can be carried out repeatedly.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; and (iii) polymerizingmeans for synthesizing nucleic acid copies of the nucleic acid analytes,such polymerizing means comprising a first set of primers, a second setof primers and a third set of primers wherein the third set comprises atleast one production center; and b) contacting the library of nucleicacid analytes with the first set of primers to form a first set of boundprimers; c) extending the first set of bound primers by means oftemplate sequences provided by the nucleic acid analytes to form firstcopies of the analytes; d) contacting the extended first copies with thesecond set of primers to form a second set of bound primers; e)extending the second set of bound primers by means of template sequencesprovided by the extended first copies to form second copies of thenucleic acid analytes; f) contacting the second copies with the thirdset of primers to form more than one third bound entity to form a thirdset of bound primers; g) extending the third set of bound primers bymeans of template sequences provided by the extended second set ofprimers to form a hybrid comprising a second copy, a third copy and atleast one production center; h) synthesizing from the production centerin the second set of primers in the complexes one or more nucleic acidcopies under isothermal or isostatic conditions; i) hybridizing thenucleic acid copies formed in step i) to the array of nucleic acidsprovided in step a) (i); and j) detecting or quantifying any of thehybridized copies obtained in step i). Elements recited in this processand variations and subelements are as described elsewhere in thisdisclosure. Of special mention is the use of random primers as thesecond set of primers. Furthermore, the second set of primers can becomplementary to the primer binding site where the process comprises anadditional step c′) of including a primer binding site after carryingout step c). The primer binding site can be added by means of T4 DNAligase or terminal transferase. Other aspects or variations of thisprocess can be made or carried out. The further step of separating theextended second set of primers obtained from step f) from theirtemplates and then repeating step e) can be made. Step g) can also becarried out repeatedly. An additional step f′) of separating theextended second set of primers obtained in step e) can be carried out.Also, the step of separating the first copies obtained from step c) fromtheir templates and then repeating step b) can be carried out. Further,the step of separating the extended second set of primers obtained fromstep f) from their templates and then repeating step e) can be carriedout. Step g) can also be carried out repeatedly. In another variation ofthis process, the second set of primers can comprise at least oneproduction center which differs in nucleotide sequence from theproduction center in the third set of primers.

Still another process for detecting or quantifying more than one nucleicacid of interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; and (iii) polymerizingmeans for synthesizing nucleic acid copies of the nucleic acid analytes,such polymerizing means comprising a first set of primers and a secondset of primers, wherein the first set of primers are fixed orimmobilized to a solid support, and wherein the second set of primerscomprises at least two segments, the first segment at the 3′ endcomprising random sequences, and the second segment comprising at leastone production center; (iv) means for synthesizing nucleic acid copiesunder isothermal or isostatic conditions; b) contacting the library ofnucleic acid analytes with the first set of primers to form more thanone first bound entity; c) extending the bound first set of primers bymeans of template sequences provided by the nucleic acid analytes toform first copies of the analytes; d) contacting the extended firstcopies with the second set of primers to form more than one second boundentity; e) extending the bound second set of primers by means oftemplate sequences provided by the extended first copies to form morethan one complex comprising extended first copies and extended secondset of primers; f) synthesizing from a production center in the secondset of primers in the complexes one or more nucleic acid copies underisothermal or isostatic conditions; g) hybridizing the nucleic acidcopies formed in step f) to the array of nucleic acids provided in stepa) (i); and h) detecting or quantifying any of the hybridized copiesobtained in step g). The above-recited elements and variations andsubelements thereof have been described elsewhere and previously in thisdisclosure. Those descriptions apply equally to this process.

Another significant process worth discussion is one for detecting orquantifying more than one nucleic acid of interest in a library. Thisprocess comprises the steps of a) providing (i) an array of fixed orimmobilized nucleic acids identical or complementary in part or whole tosequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers,wherein the first set of primers are fixed or immobilized to a solidsupport, and wherein the first set of primers comprise at least oneproduction center; and (iv) means for synthesizing nucleic acid copiesunder isothermal or isostatic conditions; b) contacting the library ofnucleic acid analytes with the first set of primers to form more thanone first bound entity; c) extending the bound first set of primers bymeans of template sequences provided by the nucleic acid analytes toform first copies of the analytes; d) extending the first copies bymeans of at least four (4) or more non-inherent homopolymericnucleotides; e) contacting the extended first copies with the second setof primers to form more than one second bound entity; f) extending thebound second set of primers by means of template sequences provided bythe extended first copies to form more than one complex comprisingextended first copies and extended second set of primers; g)synthesizing from a production center in the second set of primers inthe complexes one or more nucleic acid copies under isothermal orisostatic conditions; h) hybridizing the nucleic acid copies formed instep g) to the array of nucleic acids provided in step a) (i); and i)detecting or quantifying any of the hybridized copies obtained in steph). The elements recited above in this process and variations andsubelements are described elsewhere in this disclosure. Thosedescriptions apply to this process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; (iii) polymerizing meansfor synthesizing nucleic acid copies of the nucleic acid analytes, suchpolymerizing means comprising a first set of primers and a second set ofprimers, wherein the first set of primers are fixed or immobilized to asolid support, and wherein the first set comprises at least oneproduction center; (iv) a set of oligonucleotides or polynucleotidescomplementary to at least one segment or sequence of the second set ofprimers; and (v) means for ligating the set of oligonucleotides orpolynucleotides (iv); b) contacting the library of nucleic acid analyteswith the first set of primers to form more than one first bound entity;c) extending the bound first set of primers by means of templatesequences provided by the nucleic acid analytes to form first copies ofthe analytes; d) ligating the set of oligonucleotides or polynucleotidesa) (iv) to the 3′ end of the first copies formed in step c) to form morethan one ligated product; e) contacting the ligated product with thesecond set of primers to form more than one second bound entity; f)extending the bound second set of primers by means of template sequencesprovided by the ligated products formed in step d) to form more than onecomplex comprising the ligated products and the extended second set ofprimers; g) synthesizing from a production center in the second set ofprimers in the complexes one or more nucleic acid copies underisothermal or isostatic conditions; h) hybridizing the nucleic acidcopies formed in step g) to the array of nucleic acids provided in stepa) (i); and i) detecting or quantifying any of the hybridized copiesobtained in step h). Descriptions for any of the above-recited elementsin this process are given elsewhere in this disclosure, and need not berepeated except to say that such are equally applicable to this process.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; (iii) polymerizing meansfor synthesizing nucleic acid copies of the nucleic acid analytes, suchpolymerizing means comprising a first set of primers and a second set ofprimers, wherein the first set of primers are fixed or immobilized to asolid support, and wherein the second set comprises at least oneproduction center; (iv) a set of oligonucleotides or polynucleotidescomplementary to at least one segment or sequence of the second set ofprimers; and (v) means for ligating the set of oligonucleotides orpolynucleotides (iv); b) contacting the library of nucleic acid analyteswith the first set of primers to form more than one first bound entity;c) extending the bound first set of primers by means of templatesequences provided by the nucleic acid analytes to form first copies ofthe analytes; d) ligating the set of oligonucleotides or polynucleotidesa) (iv) to the 3′ end of the first copies formed in step c) to form morethan one ligated product; e) contacting the ligated product with thesecond set of primers to form more than one second bound entity; f)extending the bound second set of primers by means of template sequencesprovided by the ligated products formed in step d) to form more than onecomplex comprising the ligated products and the extended second set ofprimers; g) synthesizing from a production center in the second set ofprimers in the complexes one or more nucleic acid copies underisothermal or isostatic conditions; h) hybridizing the nucleic acidcopies formed in step g) to the array of nucleic acids provided in stepa) (i); and i) detecting or quantifying any of the hybridized copiesobtained in step h). For a description of the elements recited in thisprocess, refer to any of the several preceding paragraphs.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical or complementaryin part or whole to sequences of the nucleic acids of interest; (ii) alibrary of nucleic acid analytes which may contain the nucleic acids ofinterest sought to be detected or quantified; and (iii) polymerizingmeans for synthesizing nucleic acid copies of the nucleic acid analytes,such polymerizing means comprising a first set of primers, a second setof primers and a third set of primers, wherein the first set of primersare fixed or immobilized to a solid support, and wherein the third setcomprises at least one production center; and b) contacting the libraryof nucleic acid analytes with the first set of primers to form more thanone first bound entity; c) extending the bound first set of primers bymeans of template sequences provided by the nucleic acid analytes toform first copies of the analytes; d) contacting the extended firstcopies with the second set of primers to form more than one second boundentity; e) extending the bound second set of primers by means oftemplate sequences provided by the extended first copies to form anextended second set of primers; f) separating the extended second set ofprimers obtained in step e); g) contacting the extended second set ofprimers with the third set of primers to form more than one third boundentity; h) extending the third bound entity by means of templatesequences provided by the extended second set of primers to form morethan one complex comprising the extended third bound entity and theextended set of primers; i) synthesizing from a production center in thesecond set of primers in the complexes one or more nucleic acid copiesunder isothermal or isostatic conditions; j) hybridizing the nucleicacid copies formed in step i) to the array of nucleic acids provided instep a) (i); and k) detecting or quantifying any of the hybridizedcopies obtained in step j). See this disclosure for a discussion of anyof the above-recited elements.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto sequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; and (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers; b) contacting the nucleic acidanalytes with the first set of primers to form a first bound entity; c)extending the bound set of first set of primers by means of templatesequences provided by the nucleic acid analytes to form first nucleicacid copies of the analytes; d) separating the first nucleic acid copiesfrom the analytes; e) repeating steps b), c) and d) until a desirableamount of first nucleic acid copies have been synthesized; f)hybridizing the nucleic nucleic acid copies formed in step e) to thearray of nucleic acids provided in step (i); and g) detecting orquantifying any of the hybridized first nucleic acid copies obtained instep f). These elements are described elsewhere in this disclosure.

Another process for detecting or quantifying more than one nucleic acidof interest in a library comprises the steps of a) providing (i) anarray of fixed or immobilized nucleic acids identical in part or wholeto sequences of the nucleic acids of interest; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest sought tobe detected or quantified; (iii) polymerizing means for synthesizingnucleic acid copies of the nucleic acid analytes, such polymerizingmeans comprising a first set of primers and a second set of primers;(iv) means for addition of sequences to the 3′ end of nucleic acids; b)contacting the nucleic acid analytes with the first set of primer toform a first bound entity; c) extending the bound set of first set ofprimers by means of template sequences provided by the nucleic acidanalytes to form first nucleic acid copies of the analytes; d) extendingthe first nucleic copies by the addition of non-template derivedsequences to the 3′ end of the first nucleic acid copies; e) contactingthe extended first nucleic acid copies with the second set of primers toform a second bound entity; f) extending the bound set of second set ofprimers by means of template sequences provided by the extended firstnucleic acid copies to form second nucleic acid copies; g) separatingthe second nucleic acid copies from the extended first nucleic acidcopies; h) repeating steps e), f) and g) until a desirable amount ofsecond nucleic acid copies have been synthesized; i) hybridizing thesecond nucleic acid copies formed in step h) to the array of nucleicacids provided in step (i); and j) detecting or quantifying any of thehybridized second nucleic acid copies obtained in step i). Descriptionsfor any of the above-recited elements are provided elsewhere in thisdisclosure.

An illustrative example of this aspect of the present invention would beto bind a poly T primer to poly A mRNA and extend it by Tth DNApolymerase under conditions that allow it to be used as a ReverseTranscriptase. Thermal denaturation followed by binding of an unextendedpoly T primer would allow synthesis of another copy by Tth DNAPolymerase. The amount of amplification would be proportional to a) thenumber of primer binding sites on an individual template molecule b) theefficiency of binding/extension and c) the number of cycles carried out.Thus, with a single primer binding site in a target analyte, 50%efficiency and 100 cycles denaturation/repriming, the method of thepresent invention can produce 50 1^(st) cNA copies from a single analytemolecule.

In another aspect of the present invention, primers are used to generatea library of nucleic acids with production centers capable ofsynthesizing multiple nucleic acid copies that comprise sequences thatare either identical or complimentary to sequences in the originalanalytes. In the first step of this particular aspect of the presentinvention, the entire population or a subset of the population ofnucleic acids analytes is used to synthesize 1^(st) strand nucleic acidcopies as described previously for linear amplification. In the nextstep of this aspect of the present invention, the 1^(st) cNA strand ismade available for further binding/extension events by the removal ordestruction of the template strands. This can be carried out by avariety of physical, chemical and enzymatic means. Examples of suchmethods can consist of but not be limited to denaturation, alkali orRNase treatments. Denaturation can be carried out by exposure to highheat or by the other methods described above for multiple cycles oflinear amplification, thereby allowing them to participate in latersteps. In the next step, primers are annealed to the 1^(st) cNA strandin order to synthesize the complementary strands, thereby generatingdouble-stranded cNA copies of the original analyte population. Theprimers used for 2^(nd) strand synthesis are designed such that their 5′ends comprise sequences capable of acting as production centers. Adescription of such production centers is disclosed in Rabbani et al.,U.S. patent application Ser. No. 08/574,443, filed on Dec. 15, 1995(Novel Property Effecting And/Or Property Exhibiting Compositions forTherapeutic and Diagnostic Uses), abandoned in favor of U.S. patentapplication Ser. No. 08/978,632, filed on Nov. 25, 1997), incorporatedherein by reference. An example of a production center that would beparticularly useful in the present invention would comprise an RNApromoter segment.

For example, random hexamer primers for 2^(nd) strand synthesis can havethe structure:

-   -   5′-promoter-N₁N₂N₃N₄N₅N₆-3′        In a preferred mode, the promoter is a phage promoter. The        sequences specific for their cognate polymerases are        sufficiently short that their addition onto an oligounucleotide        being used for priming allows synthesis to remain both efficient        and inexpensive. At the same time, they are sufficiently long        that they are unique compared to the genomic DNA they are being        used with. Also, the phage RNA polymerases that recognize these        promoters are usually single protein molecules that have no        requirement for other subunits or cofactors. Of special use in        this aspect of the present invention are phage promoter        sequences that are recognized by the T3, T7 and SP6 RNA        polymerases. These enzymes are well characterized and are        commercially available from a number of sources.

For efficient functionality, the promoters cited as examples aboveshould be in double-stranded form. This may be carried out in severaldifferent ways. A potential sequence of events for one such method isgraphically depicted in FIG. 4. If the polymerase used for extension hasstrand displacement activity, the primer binding closest to the 3′ endof the 1^(st) strand (Primer A in FIG. 4) remains bound to the template,but the other extended primers (Primer B and Primer C) are released fromthe template in single stranded form. Thus, a given individual templatemolecule may give rise to a plurality of complementary copies bymultiple priming/extension events with two groups of products:essentially double-stranded molecules that comprise the 1^(st) cNAstrands bound to their complements and single-stranded molecules derivedfrom the displaced strands.

Although initially the displaced strands are in single-stranded form,the continued presence of other primers from either 1^(st) or 2^(nd)strand synthesis could allow further binding/extension events thatconvert the displaced single strands into double-stranded form.Alternatively, there may have been intermediary purification steps takento separate extended primers from non-extended primers. For example,separation may be useful to minimize or prevent the synthesis ofmolecules with promoters at each end. Such double-ended constructs maynot transcribe efficiently or may produce nucleic acids that hybridizewith each other rather than the target elements of the array. Therefore,the same primers that were used to initiate synthesis of the 1^(st) cNAstrand can be added to the mixture with the displaced 2^(nd) cNA strandsas well as whatever reagents may also be necessary to convert thedisplaced single-stranded DNA molecules into double-stranded products.Alternatively, random primers without promoters may be used for primingthe displaced 2^(nd) cNA strands. The synthesis of a complementary copyfor the displaced single strands also converts the promoter segment inthe 5′ end of these molecules into double-stranded form.

On the other hand, the promoter in the extended primer that remainsbound to the original 1^(st) cNA strand template (Primer A in FIG. 4)needs different processes to render it into a functionally efficientform. For instance, the single-stranded 3′ tail of the 1^(st) cNA strandcould be digested by the 3′ to 5′ Exonuclease activity of T4 DNApolymerase. Upon reaching the double stranded portion, the enzyme couldthen use its polymerase activity to extend the shortened 3′ end by usingthe promoter segment of primer A as a template thereby generating adouble-stranded promoter. In another approach, oligonucleotides can beprovided that are complementary to the single-stranded promotersequences (FIG. 5 a) or the primers used for 2^(nd) strand cNA synthesiscan be designed such that they are self-complementary and form stem loopstructures that generate double-stranded functional promoters (FIG. 5b). Lastly, the 2^(nd) cNA strands bound to the template can bedenatured and the same processes described above for converting thedisplaced 2^(nd) cNA strands can be used to convert them intodouble-stranded form.

The creation of functional transcriptional units from the originaldiverse nucleic acid analytes allows amplification by making multipletranscript copies from each cNA template. By inclusion of the RNApromoter sequence in primers that used the 1^(st) cNA strand as atemplate, all the resultant transcripts are also complementary to the1^(st) cNA strand. However, some target arrays that use definedoligonucleotide sequences as target elements have been designed for thepurpose of detecting labeled 1^(st) cDNA copies of mRNA rather thantheir complements. In such a case, the transcription products of theseries of reactions described above can be used as templates tosynthesize sequences equivalent to labeled 1^(st) cDNA copies by reversetranscription. As described previously, random or selected primers mayfind use for this purpose. This conversion step may offer otheradvantages as well since DNA is known to be more stable than RNA and hasrelatively less secondary structure compared to RNA.

RNA transcripts or cDNA copies of the RNA transcripts created from theprocesses described above can either be labeled or unlabeled. When thepolynucleotides are unlabeled, they can use UDTs for signal generation.As described previously, the original anlytes may have inherent UDTsequences that may serve this function or the analytes may be modifiedby the incorporation of non-inherent UDT sequences. On the other hand,the synthetic steps that are carried out in the series of reactionsabove provide the opportunity to incorporate non-inherent UDTs duringeither 1^(st) strand or 2^(nd) strand synthesis by primers withappropriate designs. For example, a primer design for 2^(nd) strandsynthesis can have the following structure:

-   -   5′ promoter-UDT-hexamer-3′.

After binding the primer above to a 1^(st) cNA strand followed byextension, the transcripts could be generated with the structure:

-   -   5′ UDT-hexamer-RNA sequence-3′.        Although the transcript shown above has a UDT at its 5′ end,        other designs allow the transcripts to be synthesized with UDTs        in their 3′ ends. For instance, this can take place by either        the sequence of the primer binding site used for the initial        1^(st) strand synthesis being capable of acting as a UDT or by        incorporation of a UDT into the primer that is to be used for        1^(st) strand synthesis. As an example of both methods, a        transcription unit can be synthesized from poly A RNA by priming        of the 1^(st) cNA strand with an oligonucleotide primer with the        structure:    -   5′ UDT OligoT-3′        and priming of the 2^(nd) cDNA strand by an oligonucleotide        primer having the structure    -   5′ promoter-hexamer-3′.        The double-stranded product of 1^(st) cNA and 2^(nd) cNA strand        synthesis reactions would then have the following structure:    -   5′ promoter-hexamer-2^(nd) strand sequence-PolyA-UDT 3′        Transcription from this construct would generate RNA molecules        that have the following structure:    -   5′ hexamer-2^(nd) strand sequence-PolyA-UDT 3′        The product above can bind a UDE either through the an inherent        UDT (the Poly A sequence) or through the artificially        incorporated UDT. In addition, it should be recognized that the        incorporation of UDTs for signal generation can be coupled with        incorporation of labeled nucleotides if desired. Thereby, either        by direct labeling or by the presence of UDTs, this aspect of        the present invention provides for the synthesis of a library of        detectable products that will reflect the initial levels of the        various nucleic acid analytes of a library.

The use of amplification utilizing RNA synthesis has been previouslydescribed by Kwoh and Gingeras, (1989, Proc. Nat. Acad. Sci. USA 86;1173-1177; incorporated herein by reference) but the purpose of thatwork was in diametric opposition to the present invention. In Kwoh andGingeras, primers with specific sequences were used to synthesize the2^(nd) cDNA strand in order to amplify a single defined discretesequence that was of interest. Thus there is no suggestion orrecognition of potential benefits of amplification of a diversepopulation of various nucleic acids.

In a patent application that was filed in the same year as thepublication by Kwoh and Gingeras, a method was described by van Gelderet al. (U.S. Pat. No. 5,716,785; incorporated herein by reference) forlinear amplification of a general population of RNA targets by includinga phage promoter into the primer used for the 1^(st) cDNA strand.Synthesis of the 2^(nd) strand were carried out either by nicking of theRNA template by RNase H or by hairpin formation at the end of the 1^(st)cDNA strands to provide self-priming events. Furthermore, the claims forthis patent and a related patent by the same inventors (U.S. Pat. No.5,891,636; incorporated herein by reference) specifically includes thephrase “without using an exogenous primer”. Thus, in these patents thereis firstly a requirement of inclusion of a promoter sequence into theprimers used for 1^(st) strand synthesis. Secondly there is noappreciation for the use of primers being added to catalyze the 2^(nd)strand synthesis. In fact, there is even a teaching away from thislatter concept. In addition, all of the foregoing methods synthesizeincomplete copies of the primary analytes as the completeness of thecopies made by RNase H are dependent upon the distance of the nick thatis closest to the 5′ end of the mRNA, only a minority will haverepresentation of the sequences closest to the 5′ end of the mRNA. Inaddition, there would never be representation of the end itself since itwould be used for retaining the RNA fragment/primer closest to the 5′end. Synthesis by means of hairpin formation also has intrinsicallyincomplete representation of the 5′ end sequences since nucleasedegradation of these sequences takes place during elimination of thehairpin. Also, there may be other losses since even nucleases that areconsidered to be single strand specific are more accuratelycharacterized as having a preference for single-strands since it is wellknown that there is also some level of activity with segments that arein double-stranded form.

The present invention is in contrast to previously cited art that didnot use primers for 2^(nd) strand synthesis. These methods of previousart depended upon the presence of RNaseH to create a second strand orelse required self-priming events by a foldback mechanism and subsequenttreatment with S1 nuclease or its equivalent. In the absence of such anuclease treatment, transcripts made from hairpin derived constructswould be self-complementary and thus incapable of appreciablehybridization to arrays. In contrast to this prior art, the presentinvention discloses various methods where exogenous primers are used tosynthesize the 2^(nd) strand. Also, in some aspects of the presentinvention, the methods used to synthesize the 2^(nd) strand includemeans that selectively retain information from the 5′ ends of analytes.In addition, the present invention describes the potential for thesynthesis of multiple transcription units from a single 1^(st) strandcNA template thereby providing an additional level of amplification.

It is another aspect of the present invention that the 1st cNA strandscan be actively prevented from creating 2^(nd) cNA strands through afold-back mechanism by blocking the extendability of a 1^(st) cNAstrand. One method of carrying this out is by the addition of adideoxynucleotide to the 3′ terminus of a 1st cNA copy by terminaltransferase. Although this method would prevent a 1^(st) cNA strand fromparticipating in self-priming reactions, a blocked 1^(st) can strandwould retain its capability of being used as a template. In this aspectof the present invention, either the primer used for 1^(st) strand cNAsynthesis or 2^(nd) strand cNA synthesis can comprise an RNA promoter orother replication center.

Another aspect of the present invention discloses the addition orincorporation of artificial primer binding sites to carry out the novelprocesses described above. For instance, the translation of mRNA into acDNA copy also frequently includes the terminal addition of a fewnon-template directed nucleotides into the 3′end of the 1^(st) cNAstrand by Reverse Transcriptase. In previous art, these added bases havebeen used as primer binding sites for cloning of full length cDNAmolecules. The addition of a few Cytosine nucleotides at the end of amolecule has been sufficient for the binding and extension of a primerthat has 3 Guanosine nucleotides at it 3′ end (user Manual for SMARTcDNA Technology, Clontech Laoboratories, Inc., Palo Alto, Calif.). Inthis system, aborted or stalled cDNA sequences that were incompletecopies of the original mRNA molecules would not be substrates for theaddition reaction by Reverse Transcriptase. This provided for a morecomplete representation of the 5′ sequences of the original mRNA in alibrary of cDNA clones.

The non-template derived addition of Cytosine nucleotides to the 1^(st)cDNA strand has been previously used in the process of making atranscription library (Wang et al. 2000, Nature Biotechnology 18;457-459; incorporated herein by reference). However, this system wasbasically similar to the method described by van Gelder et al., (op.cit.) since a phage promoter was included in the primers used forsynthesis of the 1^(st) cDNA strand. As such, this arrangement has thelimitation that it has lost the selectivity for molecules that havecopied completely their mRNA templates. Primers that bind to interiorpoly C sequence and initiate extensions are as competent as bindings topoly C's at the end of cDNA (Matz et al., 1999) to synthesize 2^(nd)cDNA strands, thereby creating functional double stranded phagepromoters.

In contrast to van Gelder et al., and Wang et al., this particularaspect of the present invention provides a promoter in the primer usedfor the 2^(nd) strand synthesis. Thus, the novel processes that havebeen disclosed previously can be carried out by the use of a primer for2^(nd) strand synthesis that comprises oligo dG sequences at their 3′end for binding to the termini of 1 cNA strands. In this aspect of thepresent invention, priming events that derive from the terminal bindingsand extensions will lead to double stranded promoters in molecules. Asillustrated in Step (D) in FIG. 6, a primer with a T7 promoter can bindto the terminus of the 1^(st) cNA strand. Extension of this primer cancreate a double stranded molecule where the 3′ end of the primer isextended using the cDNA as a template and the 3′ end of the cNA isextended using the primer sequences as a template. The net product ofsuch extensions would be a double stranded transcription unit. On theother hand, Step (E) of FIG. 6 shows the binding of a primer with a T7promoter to an internal segment of the cNA with. In this case, althoughthere can be extension from the 3′ end of the primer to create apartially double-stranded molecule, the 3′ end of the cNA is unable touse the primer as a template, thus leaving the promoter in anon-functional single-stranded form.

One advantage of the system described above is that the non-templateaddition of nucleotides can be carried out by enzymes that are alreadypresent in the reaction mixture. On the other hand, if desired, TerminalTransferase can be added to increase control over the reaction andimprove efficiency. When poly A, T or U sequences are already present ineither RNA, DNA or cNA copies, it is preferred that the Terminaltransferase use dGTP or dCTP. Primers for 2^(nd) strand synthesis canthen be designed whose sequences comprise a promoter and a 3′ segmentcomplementary to the sequences added by the Terminal Transferaseaddition step. The steps of this process are shown in FIG. 7, wheresubsequent extensions to create a double stranded promoter can becarried out as previously described for FIG. 6. Also, since the directedaddition of nucleotides takes place only where there is either a doublestranded end or a free 3′ end, only cDNA molecules that have beencompletely extended to the ends of the analyte templates will besuitable substrates for terminal addition.

Since these additions can be longer than those derived from non-templateadditions by Reverse Transcriptase, the primers used for 2^(nd) strandsynthesis can have longer corresponding homopolymeric segments therebyallowing higher temperatures for binding and extension. This heightenedstringency should decrease the frequency of priming events with internalsequences in the 1^(st) cNA template strand and provide higherrepresentation of sequences from the 5′ end of the original analytes.Therefore, when terminal transferase is used to generate a primerbinding site for 2^(nd) strand synthesis, the promoter can be in eitherthe 1^(st) strand or the 2^(nd) strand. The step of terminal transferaseaddition to the 1^(st) cNA can be carried out while it is still bound toits template as described above, or it can be carried out afterdestruction of the template or separation of the template from the1^(st) cNA strand. This method should continue to enjoy 2^(nd) strandsynthesis that is preferentially initiated by primers binding and beingextended from the 3′ termini of 1^(st) cNA strands. As describedpreviously, UDTs, as well as labeled or unlabeled nucleotides can all beutilized in carrying out this aspect of the present invention. Also, itis contemplated that higher yields of end products can be achieved byrepetitions of one or more steps of the various process that aredisclosed herein.

Other means that preferentially carry out priming events at the 3′ endsof 1^(st) strand cNA's may also find use in the present invention. Forinstance, a cDNA copy that is a complete copy of its RNA template is asubstrate for blunt end ligation by T4 DNA ligase with a double-strandedoligonucleotide. The sequence of the oligonucleotide ligated to the 3′end of the 1^(st) cNA strand can be chosen by the user and can functionas a primer binding site for making a 2^(nd) cNA strand. Similarly a 3′single-stranded tail in the 1^(st) cNA strand is a substrate forligation of a single-stranded DNA oligonucleotide by T4 RNA ligase(Edwards et al., 1991 Nucleic Acids Research 19; 5227-5232; incorporatedherein by reference). Lastly, a double-stranded oligonucleotide with a3′ single-stranded tail can be joined to a 1^(st) strand cNA through“sticky end” ligation by T4 DNA ligase when the 1^(st) cNA andoligonucleotide tails are complementary. As described previously, thesecNA tails can be derived from non-template additions by ReverseTranscriptase or by Terminal transferase. Illustrative examples of theseprocesses are given in FIG. 8. Since all of these processes aredependent upon preferential binding of primers to the 3′ ends 1^(st)strand can molecules, the promoter can be in either the 1^(st) or 2^(nd)cDNA strand.

In another embodiment of the present invention, a 1^(st) strand cNAstrand is fragmented by physical, chemical or enzymatic means. Examplesof enzymatic means can include but not be limited to restriction enzymessuch as Hha I, Hin P1 I and Mnl I, DNases such as DNase I and nucleasessuch as S1 nuclease and Mung Bean Nuclease. These fragments can be usedas templates for synthesis of a 2^(nd) strand by any of the methodsdescribed previously. For example, hybridization and extension of randomprimers with T7 promoters can be used with the cNA strand fragments astemplates in processes similar to those shown in FIGS. 4 and 5. Or ifpreferred, the homopolymeric addition or ligation steps described abovecan be carried out to provide specific primer binding sites. FIG. 8 isan illustration of this process using the homopolymeric method. Breakingdown the 1^(st) strand copy into smaller segments followed byincorporation of a primer during 2^(nd) strand synthesis would providesmaller transcription units. This may be advantageous when usingmodified nucleotides for signal generation. For instance, when there arelong stretches in the template strand that are complementary to thelabeled nucleotide, the modification to the nucleotide may cause ablockage in downstream transcription or loss of processivity and resultin under-representation of those sequences. In this particular aspect ofthe present invention, the partition of copies of analyte sequences intosmaller individual transcription units allows each of the units todirect RNA synthesis independently thereby creating a more completerepresentation of the library of various nucleic acid sequences.

In another embodiment of the present invention, the novel methodsdisclosed for synthesis of a library are combined with capture methodsto provide more efficient synthesis as well as flexibility in changingsalts, buffers, enzymes and other components during multistep processes.The present invention discloses the use of a 1^(st) strand primer thatis bound to a solid matrix such as a bead followed by the processesdescribed above. For example, the 3′ end of Oligo T sequences bound to asolid matrix can be extended using polyA mRNA as a template. Inaccordance with the methods of the present invention, this 1^(st) cNAstrand is thereupon used as a template for the 2^(nd) cNA strand. Whencarrying out this aspect of the present invention, a replicative centersuch as an RNA promoter sequence can be introduced into either the1^(st) or 2^(nd) strand depending upon the particular method used. Forinstance, random primers with promoters in their 5′ ends can bind to theextended 1^(st) cDNA strands to create 2^(nd) strands that have apromoter incorporated into them. This process is depicted in FIG. 10.

The single-stranded promoter on the 5′ ends of the 2^(nd) cDNA strandscan be converted into double-stranded form by any of the methodsdescribed previously. For instance, the primer/template complex thatremains bound to the bead in FIG. 10 can be treated with T4 DNApolymerase, hybridized with an oligonucleotide complementary to thepromoter segment or the primer can be designed with self complementaryregions. The latter two methods were previously discussed with referenceto FIG. 5. With regard to the displaced 2^(nd) cDNA strands in FIG. 10,the presence of unextended oligo-T tails on the matrix material canprovide further binding/extension events since the displaced strandscarry poly A sequences on their 3′ ends. However, if preferred, moreoligo-T can be added whether associated with beads or free in solution.Extension of the oligo-T should ultimately result in conversion of thesingle-stranded promoters of the displaced 2^(nd) cDNA strands intofunctional double-stranded forms.

Another method that can be used in the present invention is to repeatone or more of the steps that have been described in the presentinvention. For instance, after using a library of analytes to synthesize1^(st) can copies attached to a matrix, the anlytes can be separatedfrom the 1^(st) cNA copies and used to create another pool of 1^(st) cNAcopies. Similarly, after synthesis of 2^(nd) can strands, the library of2^(nd) cNA strands can be separated from the 1^(st) can strands fixed tothe matrix. All 2^(nd) cNA strands that have copied the 5′ ends of the1^(st) cNA strands will have regenerated the sites that were initiallyused to bind to the primers linked to the beads. If desired, the 2^(nd)strands can be rebound to the same beads. Since there are likely to bean enormous number of poly T primers on the beads compared to the numberof templates used for 1^(st) cNA synthesis, the majority of primers onthe matrix remain unextended and can be used for new priming events.Thus, complete copying of these rebound 2^(nd) can strands should allowgeneration of double-strand promoters at the ends of these moleculeswithout a necessity for the use of T4 to do “trimming”. If desired the1^(st) cNA strands that are attached to the matrix can be used togenerate another pool of 2^(nd) cNA strands. The pool or pools of 2^(nd)can strands can then be added to fresh beads with primers complementaryto their 3′ ends. Again, the extension of the primers attached to thematrix will convert all of the 2^(nd) can strands into double-strandedform including the promoter sequences that were at their 5′ ends.Lastly, after a transcription reaction is carried out, the reactionproducts can be removed and the nucleic acid on the matrix can be usedfor more transcription reactions thereby accumulating more transcriptionproducts.

Although the example above describes priming of an analyte with a poly Asegment by an oligo T primer attached to a matrix, thee primers can alsobe prepared with one or more discrete bases at their 3′ ends. Asdescribed previously, these primers can be used as a group thatrepresents all the possible variations or they can be used individuallydepending upon whether general amplification or separation intosubclasses was desired. The poly A sequence used above is understood toonly be an illustrative example. As described previously, the sequencesin analytes used for binding of 1^(st) strand primers can be derivedfrom inherent sequences or they may be noninherent sequences in analytesthat have been artificially introduced by any of the means that havebeen described previously. This particular embodiment of the presentinvention can utilize any of these primer binding sites by appropriatedesign of the primer sequence bound to the matrix.

In the present invention, the primer sequences for 1^(st) strandsynthesis can be either directly or indirectly attached to a matrix.Methods for direct attachment of oligonucleotides to matrixes are wellknown in the art. In addition, beads with covalently attached extendablepoly T segments are commercially available from a number of sources.Methods for indirect attachment are also well known in the art. Forinstance FIG. 11 depicts a sandwich method where a primer has twosegments, one of which is complementary to a capture segment attached tothe matrix and the other is complementary to the poly A segment of thetarget RNA. The two segments of the primer may form a continuousnucleotide sequence or there may be a disjunction between the twosegments. Hybridization of the two segments of the primer and thecomplementary sequences on the matrix and the binding site of theanalyte can take place simultaneously or they can be carried out in astep-wise fashion. For instance, hybridization of target RNA to thecapture element can be carried out in solution followed by capure to thematrix. It is preferred that the segment that is bound to the matrix berendered incapable of extension. One way this blockage can be carriedout is by the use of the 3′ end as the attachment point to the matrix asdepicted in FIG. 11. Binding and extension events can take place asdescribed previously for FIG. 10 to synthesize 1^(st) and 2^(nd) cDNAcopies of the original poly A mRNA. Conversion of the promoter sequencesinto double-stranded form can also take place as described above.Transcription can take place either while the transcription units areattached to the matrix or if desired separation from the matrix can takeplace in a step subsequent to the transcription.

Incorporation of an RNA promoter during 1^(st) strand synthesis resultsin transcripts that comprise sequences that are complementary tosequences in the original analytes. Incorporation of an RNA promoterinto the 2^(nd) strand synthesis results in the production oftranscripts that comprise sequences that are identical to sequences inthe original analytes. As described previously, these can easily beconverted into complementary cDNA copies if desired.

It is a further subject of the present invention that transcriptionunits can be synthesized without incorporating a promoter sequence intoeither the 1^(st) cNA (as described by Eberwine et al., op. cit.) or the2^(nd) cNA strand (as described in previous embodiments of the presentinvention). As shown in step D of FIG. 12, when using extended 1 cNAstrands as templates for synthesis of the 2^(nd) cNA strands, aduplicate of the original primer binding sequence is synthesized. Thus,in FIG. 12 a.polyA segment is created at the 5′ ends for both displaced2^(nd) cNA strands and for 2^(nd) cNA strands that remain bound to thebeads. After removing these 2^(nd) cNA strands, oligonucleotide primerscomprising an RNA promoter and oligo-T sequences can be hybridized tothe 2^(nd) cNA strands. The primers may be attached to a matrix or theymay be free in solution. Provision of DNA Polymerase, nucleotides andappropriate cofactors can allow extension of both the 3′ ends of thepromoter/primers as well as the 3′ ends of the cDNA copies therebycreating functional transcriptional units as shown in step F of FIG. 12.Transcription from these DNA molecules will result in products thatcomprise sequences that are complementary to sequences in the originalanalytes.

In previous art the most common use of oligo-T that is attached to amatrix such as cellulose or beads has been for the purpose of aselective isolation of polyA mRNA followed by a release step prior tosynthesis of a library. In one instance, a special oligo T primer joinedto a T7 promoter was extended using RNA as template to create a library(Eberwine op.cit.). However, this system put the promoter in closeproximity to the capture bead, potentially decreasing its ability to beconverted into double-stranded form and/or for it to function as apromoter. Also, synthesis of the 2^(nd) strand by random priming doesnot prevent hairpin self-priming. In the absence of a nuclease step,transcription units would direct synthesis of self-complementary RNAsfrom hairpin template sequences that would be incapable of hybridizingto target arrays use of the templates for this non-productive synthesismay cause an inefficiency in the amount of effective labeledtranscripts.

A particular benefit of the use of promoters in primers used for 2^(nd)cNA synthesi present invention is that although 1^(st) cNA strands canbe synthesized under conditions that have the potential for self-primingevents i.e. creating 2^(nd) cDNA strands by a fold-back mechanism, theabsence of a promoter in 1^(st) cDNA; strand would prevent theseconstructs from being transcriptionally active. Thus, only 2^(nd) cDNAstrands that are derived from priming events by oligonucleotides withpromoter sequences are functional for transcription. This in contrast tothe system previously described by Eberwine (op. cit.). Contrariwise,methods have also been described in the present invention that allow theuse of a promoter in the 1^(st) strand by either preventing extension ofa 1^(st) cNA strand or by facilitating 2^(nd) strand synthesis frompriming events at the ends of 1^(st) strand templates.

It is another object of the present invention to provide a method forcomparative analysis that requires only a single RNA population to belabeled. This particular aspect takes advantage of competitive bindingby an unlabeled population of RNA. Synthesis of this material can takeplace by any of the means described in the foregoing work. Theparticular sequences can be homologous to sequences that are present onthe arrays or they may be homologous to sequences that are present inthe labeled material. By comparison of hybridization of the labeledmaterial in the presence or absence of competitor, relative levels ofincreased or decreased mRNA synthesis can be established relative to thecompetitor, ie. differential competition. Adjustments can be made in therelative amounts of unlabeled material being used or the housekeepinggenes that are present as controls can allow for normalization values.This method provides the advantage that multiple sequential or parallelhybridizations can be carried out and compared with a single commonlabeled control population of RNA.

The various steps of the present invention can be carried outsequentially by adding various reagents and incubation steps asrequired. On the other hand, the series of steps can be segregated byintroducing additional steps that either remove or inactivate componentsof the reaction or where a desired product is separated from a reactionmixture. An example of the former can be heat inactivation of ReverseTranscriptase. An example of the latter can be isolation of RNA/DNAhybrids by selective matrices. These additional steps can be carried outto either improve the efficiency of subsequent steps or for the purposeof preventing undesirable side reactions.

Although the previous examples have disclosed the utility of a phagepromoter in carrying out various aspects of the present invention, aproduction center is able to operate by other means as well. Forinstance, various means of introducing UDTs that serve as primer bindingsites have been previously described in the context of synthesis of2^(nd) copy strands followed by RNA transcription. These primer bindingsites can in themselves serve as production centers for multiple copiesof various nucleic acids under isothermal conditions.

For instance the use of primers that are designed to createtarget-dependent stem-loop structures has previously been disclosed inRabbani et al., U.S. patent application Ser. No. 09/104,067, filed onJun. 24, 1998 (Novel Processes for Amplifying Nucleic Acid,Post-Termination Labeling Process for Nucleic Acid Sequencing andProducing Nucleic Acid Having Decreased Thermodynamic Stability; forspecific isothermal amplification of selected sequences. The content ofthe aforementioned Ser. No. 09/104,067 is hereby incorporated byreference. In the present invention, UDTs can be added to the variousnucleic acids of a library to carry out the amplification disclosed inRabbani et al., U.S. patent application Ser. No. 09/104,067, cited supraand incorporated herein by reference. FIG. 13 is a depiction of a seriesof reactions that could be used to carry this out. For instance, a UDTcan be ligated to a library of poly A mRNA where the UDT comprises twosegments (termed X and Y in this Figure). In the next step, a primer(Primer 1) that comprises two segments, a poly T sequence at the 3′ endand a segment termed Z at the 5′ end is hybridized to the poly Asequences at the 3′ end of the mRNA and extended by reversetranscription to make a 1^(st) cNA copy (Steps C and D of FIG. 13) thatcontains the sequnces X′ and Y′ at the 3′ end. Removal of the originaltemplate makes the X′ segment at the 3′ end of the 1^(st) cNA copyavailable for hybridization. A second primer (Primer 2) that has twosegments, segment X at the 3′ end and segment Y′ at the 5′ end can beannealed and extended to make a 2^(nd) copy (Steps D and E) of FIG. 12.The presence of Primer 2 should also allow a further extension of the1^(st) cNA copy such that a double stranded segment is formed where theY and Y′ segments are capable of self-hybridizing and thereby creating astem-loop structure with the X and X′ segments in the loop portions asdescribed in Rabbani et al., U.S. patent application Ser. No.09/104,067, cited supra and incorporated herein by reference. Creationof a stem loop at the other end can be carried out by annealing a thirdprimer (Primer 3) which comprises two segments, segment Z at the 3′ endand a Poly A segment at the 5′ end using a 2^(nd) cNA copy as atemplate. The availablity of 2^(nd) cNA copies as templates can bederived from multiple priming events by Primer 2 at the other end (asdescribed in Rabbani et al., U.S. patent application Ser. No.09/104,067, cited supra and incorporated herein by reference, or bydenaturation of the 1^(st) and 2^(nd) strands from each other. Extensionof Primer 3 creates a structure that has the Poly T and Poly A segmentsforming a stem and the Z and Z′ segments forming the loops. Furtherbinding and extension reactions under isothermal conditions can proceedas described previously for unique targets. It should be noted that theparticular sequences used for X, Y and Z are arbitrary and can be chosenby the user. For instance, if the Z segment of Primer 1 used in step Cof FIG. 13 was designed with X and Y sequences at the 5′ end, the unitlength amplicon would have X′ and Y segments at the 3′ end of eachstrand. As such, amplification could be carried out using only Primer 2.

Another example of the use of non-inherent UDTs being used as primerbinding sites for isothermal amplification is shown in FIG. 14 for usewith the Strand Displacement Amplification system described by Walker etal., in U.S. Pat. No. 5,270,184 herein incorporated by reference. Inthis particular example, Incorporation of segment X takes place by twodifferent methods. In step B of FIG. 14, segment X is introduced byligation to an analyte of the library. In step C segment X is attachedto a poly T primer and becomes incorporated by strand extension. Thepresence of the X segment at the 5′ end of each end of the amplicon unitallows primer binding by a single Strand Displacement primer. Methodsfor the designs of primers with appropriate sequences at their 5′ endshave been described by Walker et al., (op. cit.). With regard to theparticular enzyme being used as part of the SDA system, the presence ofa particular restriction site between primer binding sites may limit theability of some sequences to be amplified in a reaction designed forgeneral amplification of a library. This may be overcome by choosingrelatively uncommon sequences or carrying out parallel reaction withdifferent enzymes.

It should be pointed out that in the examples shown in FIGS. 13 and 14,the presence of primer binding sites at each end allows exponentialamplification. However, these processes can be changed to linearamplification by designing amplicons that have binding sites forisothermal amplification at only one end of the amplicon.

Incorporation of a primer binding site that can be used for isothermalproduction of multiple copies can take place by any of the stepsdescribed previously that used a promoter in the example. For instance,FIGS. 13 and 14 show addition of an isothermal binding site directly toan analyte and also show incorporation of an isothermal binding siteduring synthesis of a first copy. FIG. 15 shows a similar situation, butin this example segment X is incorporated during 1^(st) cNA synthesis,segment Q is added after first strand synthesis and segment Z is addedduring 2^(nd) cNA strand synthesis. As described previously, one or moreof these segment can comprise primer binding sites for isothermalsynthesis. It should also be pointed out that in FIGS. 13 through 15both inherent and non-inherent UDTs were used as part of the examples.

In another aspect of the present invention, UDTs are used as primerbinding sites for amplification on an array. In this particular aspect,each locus on an array comprises two sets of primers. The first set of alocus comprises Selective Primer Elements (SPE's) that are specific fora particular analyte. The second set of a locus comprises UniversalPrimer Elements (UPE's) that are identical or complementary to sequencesin UDT elements. As described previously, UDTs can be derived fromnaturally occurring sequences or they may be artificially incorporated.The SPE″s at a locus would be able to bind to the complementarysequences in the nucleic acids of a library, thereby binding discretespecies of nucleic acids to that particular locus of the array. The useof appropriate conditions, reagents and enzymes would allow an extensionof an SPE using the bound nucleic acid as a template.

As an example of this aspect of the present invention, FIG. 16 depictsan array with three different loci termed Locus P, Locus Q and Locus R.At each of the loci, there is a set of SPE's bound to the array that arecomplementary to a particular sequence in cDNA copies made from one ofthree species of poly A mRNA termed P, Q and R respectively. Inaddition, each locus of the array in FIG. 16 has a set of UPE's thatcomprises poly T sequences. Synthesis of a cDNA copy of each of the mRNAtemplates by Poly T priming of their polyA tails creates cDNA P, cDNA Qand cDNA R respectively. Binding of the 1^(st) cDNA strand of an analyteto an SPE should be selective for each species at a particular locus. Onthe other hand, there should be little or no binding of the cDNA copiesto the universal Poly T sequences in the UPE's of the array of FIG. 16.The addition of enzymes and reagents for extension should generate2^(nd) cDNA copies of P, Q and R at the LP, LQ and LR sites on the arrayby extension of SPE's using the bound cDNA as templates. Each of these2^(nd) cDNA copies would comprise unique sequences complementary to the1^(st) cDNA strand templates. However, in addition to these uniquesequences, the 2^(nd) strand copies would include a common poly Asequence at their 3′ ends. At this stage it may be preferable to removeunhybridized analytes as well as templates used for 2^(nd) strandsynthesis. This is most easily carried out by heat denaturation followedby washing steps. The product at this stage is an array that hasextended and un-extended SPE's at each locus where the number ofextended SPE's should be in proportion to the amount of the originalcorresponding analytes. The extended SPE's can now serve as templateswhen an unextended poly T UPE is in sufficient proximity. The design andplacement of pairs of unique primers for solid phase amplification hasbeen previously described in detail in U.S. Pat. No. 5,641,658, herebyincorporated by reference. Methods for synthesis of arrays with twodifferent sequences at each locus has also been described by Gentalenand Chee, 1999 (Nucl. Acids Res. 27; 1485-1491) incorporated byreference. The same primer design rules may also be applied to thepresent invention that uses non-unique primers. Extension of a UPE witha nearby extended SPE as a template creates a new template that can inturn be used as a template for a nearby unextended SPE. This process canproceed through a series of binding and extension steps thatalternatively using SPE's and UPE's to accumulate nucleic acids that arederived from target nucleic acids homologous to the sequences in the SPEat each locus. An illustration of these steps is given in FIGS. 16through 19.

Methods for the design and synthesis of arrays for solid phaseamplification have been described in U.S. Pat. No. 5,641,658 and Weslinet al., 2000, (Nature Biotechnology 18; 199-204; both documentsincorporated herein by reference) for utilization of totally unique setsof primers. Methods of assaying the extent of synthesis are alsodescribed in these references. For example, labeled precursors can beincluded in the reaction to synthesize a labeled amplification product.Alternatively, normal precursors can be used with signal generationprovided by intercalating dyes binding to amplification products.

This invention provides unique compositions and processes for solidphase amplification. Among such compositions is one that comprises anarray of solid surfaces comprising discrete areas, wherein at least twoof the discrete areas each comprises a first set of nucleic acidprimers; and a second set of nucleic acid primers; wherein thenucleotide sequences in the first set of nucleic acid primers aredifferent from the nucleotide sequences in the second set of nucleicacid primers; wherein the nucleotide sequences of a first set of nucleicacid primers of a first discrete area and the nucleotide sequences of afirst set of nucleic acid primers of a second discrete area differ fromeach other by at least one base; and wherein the nucleotide sequences ofthe second set of nucleic acid primers of a first discrete area and thenucleotide sequences of the second set of nucleic acid primers of asecond discrete area are substantially the same or identical. Previousdescriptions for any of the above-recited elements have been givenelsewhere in this disclosure, and resort may be made to thosedescriptions in connection with this process.

A related composition of this invention is one comprising an array ofsolid surfaces comprising a plurality of discrete areas; wherein atleast two of the discrete areas each comprises a first set of nucleicacid primers; and a second set of nucleic acid primers; wherein thenucleotide sequences in the first set of nucleic acid primers aredifferent from the nucleotide sequences in the second set of nucleicacid primers; wherein the nucleotide sequences of a first set of nucleicacid primers of a first discrete area and the nucleotide sequences of afirst set of nucleic acid primers of a second discrete area differsubstantially from each other; and wherein the nucleotide sequences ofthe second set of nucleic acid primers of a first discrete area and thenucleotide sequences of the second set of nucleic acid primers of asecond discrete area are substantially the same or identical. See thisdisclosure above and below for a description of any of the elements inthis process.

Related to the last-mentioned compositions are processes for producingtwo or more copies of nucleic acids of interest in a library comprisingthe steps of a) providing (i) an array of solid surfaces comprising aplurality of discrete areas; wherein at least two of the discrete areaseach comprises: (1) a first set of nucleic acid primers; and (2) asecond set of nucleic acid primers; wherein the nucleotide sequences inthe first set of nucleic acid primers are different from the nucleotidesequences in the second set of nucleic acid primers; wherein thenucleotide sequences of a first set of nucleic acid primers of a firstdiscrete area and the nucleotide sequences of a first set of nucleicacid primers of a second discrete area differ from each other by atleast one base; and wherein the nucleotide sequences of the second setof nucleic acid primers of a first discrete area and the nucleotidesequences of the second set of nucleic acid primers of a second discretearea are substantially the same or identical; (ii) a library of nucleicacid analytes which may contain the nucleic acids of interest; (iii)polymerizing means for synthesizing nucleic acid copies of the nucleicacids of interest; b) contacting a primer of the first set with acomplementary sequence in the nucleic acid of interest; c) extending theprimer in the first set using the nucleic acid of interest as a templateto generate an extended first primer; d) contacting a primer in thesecond set with a complementary sequence in the extended first primer;e) extending the primer in the second set using the extended firstprimer as a template to generate an extended second primer; f)contacting a primer in the first set with a complementary sequence inthe extended second primer; g) extending the primer in the first setusing the extended second primer as a template to generate an extendedfirst primer; and h) repeating steps d) through g) above one or moretimes. Elements above are described elsewhere herein.

Another related process useful for detecting or quantifying more thanone nucleic acid of interest in a library comprises the steps of a)providing (i) an array of solid surfaces comprising a plurality ofdiscrete areas; wherein at least two of such discrete areas eachcomprises: (1) a first set of nucleic acid primers; and (2) a second setof nucleic acid primers; wherein the nucleotide sequences in the firstset of nucleic acid primers are different from the nucleotide sequencesin the second set of nucleic acid primers; wherein the nucleotidesequences of a first set of nucleic acid primers of a first discretearea and the nucleotide sequences of a first set of nucleic acid primersof a second discrete area differ from each other by at least one base;and wherein the nucleotide sequences of the second set of nucleic acidprimers of a first discrete area and the nucleotide sequences of thesecond set of nucleic acid primers of a second discrete area aresubstantially the same or identical; (ii) a library of nucleic acidanalytes which may contain the nucleic acids of interest; (iii)polymerizing means for synthesizing nucleic acid copies of the nucleicacids of interest; and (iv) non-radioactive signal generating meanscapable of being attached to or incorporated into nucleic acids; b)contacting a primer of the first set with a complementary sequence inthe nucleic acid of interest; c) extending the primer in the first setusing the nucleic acid of interest as a template to generate an extendedfirst primer; d) contacting a primer in the second set with acomplementary sequence in the extended first primer; e) extending theprimer in the second set using the extended first primer as a templateto generate an extended second primer; f) contacting a primer in thefirst set with a complementary sequence in the extended second primer;g) extending the primer in the first set using the extended secondprimer as a template to generate an extended first primer; h) repeatingsteps d) through g) above one or more times; and i) detecting orquantifying by means of the non-radioactive signal generating meansattached to or incorporated into any of the extended primers in stepsc), e), g), and h). Elements above are described elsewhere herein.

For many uses, the UPE's will be present on the array duringhybridization of the analyte to complementary SPE's. However, there maybe circumstances where the presence of UPE's in this step may bedeleterious. For example, binding of the diverse nucleic acids of alibrary should preferably take place only through the action of theSPE's on the array. In contrast to the example given above, there may becases where due either to the nature of the library or the choice of UPEsequences, hybridization can take place between the library and theUPE's of an array. This event could result in a loss of efficiency inthe reaction by binding of target nucleic acids to inappropriate areasof the array. For instance, the SPE's at a particular locus would beunable to use complementary nucleic acid targets as a template if thesetargets are inappropriately bound to another physical location throughbinding of UPE's. Furthermore, UPE's would be rendered non-functional bybeing extended and synthesizing nucleic acid copies that lackcomplementary to the SPE's at that particular locus.

Accordingly, it is a subject of the present invention that UPE's may beeither non-functional or absent during the initial hybridization of alibrary to the SPE's in the array. In one method of carrying this out,advantage is taken of the universal nature of the UPE's. Although eachparticular species of SPE is relegated to a specific area of the array,the UPE's are intended to be present in multiple areas of the array. Assuch, an array can be synthesized where each locus comprises a set ofSPE's and a set of chemically activated sites that are compatible withreactive groups on UPE's. After the initial hybridization of nucleicacid targets to their appropriate SPE's, the UPE's with appropriategroups can be added universally to the array by a simultaneousattachment to all of the active sites on the array. An example ofcompatible modifications that could be used in this aspect of thepresent invention could be arrays that have maleimide groups at eachlocus and UPE's that have amine groups attached to their 5′ ends.

An alternative approach is for synthesize the array with UPE's that havebeen modified such that they are temporarily unable to function. Forexample, the UPE's could be synthesized with 3′ PO₄ groups therebyblocking any potential extension reactions. After hybridization ofnucleic acids to the various SPE's of the array followed by extension ofSPE's, the nucleic acids used as templates could be removed from thereaction. After this step, the 3′ end of the UPE's could be renderedfunctional by removal of the 3′ PO₄ groups by treatment with reagentssuch as bacteriophage polynucleotide kinase or alkaline phosphatase.Thereafter, successive reactions can take place as described previously.

An alternative approach would be the use of hybridization properties ofnucleic acids. For example, the Tm of hybridization between nucleicacids is a function of their length and base composition. Therefore, theSPE's and UPE's can be designed with Tm's that are sufficientlydifferent that salt or temperature conditions can be used thatselectively allow hybridization of the nucleic acids in the sample toSPE's. The salt and temperature conditions can be altered later to allowhybridization to the UPE's on the array and carry out the appropriateseries of reactions.

Another example would be the use of competitive hybridization. Nucleicacids or their analogues can be added that are homologous to the UPE's.By either pre-hybridization or by including a high excess of suchcompetitors, the UPE's should all be occupied with the competitornucleic acids thereby allowing binding of the nucleic acids of thelibrary to SPE's only. Furthermore, the competitors can be synthesizedin such a way that even though they are bound to the UPE's they areunable to serve as templates for extension of the UPEs. Examples ofmeans that can be used for this purpose can include but not be limitedto peptide nucleic acids and oligonucleotides with multiple abasicsites. After extension of the SPE's, both the templates used forextension of SPE's and the competitor oligonucleotides bound to theUPE's can be removed concurrently rendering both the extended SPE's andUPE's available for binding to each other.

The poly A RNA in the example shown in FIGS. 16-19 made use of aninherent UDE in eucaryotic mRNA. As described previously, UDEs can alsobe added artificially either by polymerization or ligation. Forinstance, a selected arbitrary sequence can be added to the 5′ ends of alibrary of RNA analytes by the action of T4 RNA ligase. An array couldthen be used that has SPE's for unique RNA sequences and UPE's with thesame sequences as the ligated segment. After localization of the variousspecies of RNA to their appropriate location on an array, an enzymeappropriate for reverse transcription can be added as well as theappropriate buffers and reagents to extend the SPE's therebysynthesizing 1^(st) strand cDNA copies linked to the array. Removal ofthe RNA template would then allow the complement of the UPE in the cDNAcopy to bind to a nearby UPE on the array followed by a set of reactionsas described previously. Since the choice of sequences for artificiallyadded UPE's is of arbitrary nature, this aspect of the present inventioncan be applied to a simultaneous assay of different pools of analytes byadding different discrete UPE sequences to each library. In contrast tothis, the prior art cited above makes no provision for distinguishingbetween collection of analytes from different sources that have the samesequences. An illustration of an array that could be used for thispurpose is given in FIG. 20 where two libraries are being compared. Onelibrary has been prepared by joining sequences for UPE 1 to the nucleicacids and a second library has been prepared that has sequences for UPE2 joined to the nucleic acids. It should be noted that in FIG. 20, Locus1 of the array has the same SPE's as Locus 9 but they differ in theidentity of the UPE where UPE 1 is at Locus 1 and UPE 2 is at locus 9.This is also true for Locus 2 compared to Locus 10 and so on. Thus,binding of the same sequence can take place at either Locus 1 or Locus9, but the extent of amplification that will take place at each locuswill be dependent upon the amount of bound material that contains theappropriate UPE sequence.

In addition, although the examples above have used RNA or cDNA copies aslibraries for this aspect of the present invention, it has beenpreviously disclosed that DNA may also be the initial analyte. As anexample of this aspect of the present invention, DNA can be digestedwith a restriction enzyme to create a library of fragments. Adouble-stranded UDE can then be ligated to these fragments by the actionof T4 DNA ligase. The ligated products can then be denatured andhybridized to an array of SPE's. For example, to investigate potentialSNP's at a site “X” oh a target nucleic acid, sets of SPE's can bedesigned that differ by a single nucleotide at their 3′ ends. Thesubsequent efficiency of extensions would then be dependent on how wellthe nucleotide at site “X” of the target template matched the 3′ base ofthe SPE. As an internal control, a set of SPE's can be designed thatwill utilize each strand at site “X” thereby duplicating theinformation. This process is illustrated in FIG. 21. In this particularexample, it is preferred that binding between the nucleic acid and theUPE on the array be prevented since the ligated fragments will havesequences complementary to the UPE's. Examples of means that can be usedto carry this out have been described previously whereby UPE's areabsent or non-functional during hybridization of the nucleic acids tothe SPE's. On the other hands, the nucleic acids that are being analyzedcan be treated such that sequences that are complementary to UPE's areremoved. For instance, after the ligation step described above, nucleicacids can be treated with a 3′ to 5′ double-stranded Exonuclease. Thisshould selectively remove sequences complementary to the UPE's whileretaining sequences that are identical to sequences in the UPE's.Regeneration of the sequence complementary to the UPE should then takeplace only after extension of an SPE. Also as disclosed above, the useof artificial addition of UPE sequences allows the simultaneous analysisof different pools by a selective choice of different UPE sequences foreach pool.

It is a further intent of the present invention that rather thanchoosing specific sequences derived from prior sequence information, ageneral array can be made that offers complete representation of allpossible sequences. For instance, a library of SPE's that are 4 bases inlength with permutations of all 4 variable bases would comprise 4×4×4×4distinct sequences, i.e. a total of 256 permutations. With a complexityof all potential octamer oligonucleotides with the four variable bases,there would be a total of 256×256 for a total of 65,536 permutations. Inprior art, an array covering all the possible amplification productswould require two unique primers for each individual amplification.Thusly, there would be a requirement for a total of 65,536×65,536 for atotal of 4.3×10⁹ permutations for pairs of unique octamer primers on thearray. Such high numbers may be too expensive or too complex to havepractical application. On the other hand, the present inventionovercomes this limitation by virtue of the use of UPE's. Accordingly,only the SPE's need to encompass all the possible octamer sequenceswhich results in a requirement for a total of 65,536 differentsequences, a number that is easily within the ability of currenttechnology. The number of different nucleic acid that will be amplifiedat each locus will depend upon the complexity of the library of nucleicacids applied as templates as well as the conditions used for carryingout amplification. The degree of complexity of the array can also bealtered by increasing or decreasing the number of nucleotides comprisingthe SPE's. Conversely, it has previously been pointed out that a degreeof differentiation can be achieved by adding one or more discrete basesto the UPE. For example, the use of a single variable nucleotide at theend of a polyT UPE would decrease the complexity of the analytes in alibrary that could be amplified since on average, only one out of threeof the various diverse nucleic acid analytes bound to SPE's would beable to carry out strand extension. On the other hand, the inclusion ofall 3 sets of UPE's that each carries one of the 3 potential bases incombination with complete representation of octamer SPE's would increasethe complexity of arrays from 65,536 sequences to a total of 1.97×10⁵(3×65,536) permutations. By using variable nucleotides in the last twonucleotides at the 3′ end of the UPE on an array with SPE octamers, thecomplexity would be 8.0×10⁵ (12×65,536) permutations. It also should beunderstood that the complexity of the array can have an incompleterepresentation of all potential SPE sequences. For instance, octomersthat have Tm's that are much higher or lower than the average Tm of arandom population may be not be desired to be present. Also, octamersthat have self-complementary 3′ and 5′ ends may exhibit poor bindingability. When more than one species of UPE is being used, this aspectcan be carried out with amplification carried out simultaneously witheach UPE. More preferably, reactions are carried out in parallel with agiven UPE on an array for each set of reactions.

In another aspect of the present invention, a mixed phase amplificationis carried out where SPE's at fixed locations on an array are used for1^(st) strand synthesis. but the primers used for synthesis of 2^(nd)strands are not attached to the matrix of the array. In this aspect ofthe present invention, a pool of primers for 2^(nd) strands in solutioncan make use of normal nucleic acid kinetics to find 1^(st) strandtemplates fixed to distinct loci on an array for 2^(nd) strand primingevents.

FIGS. 22-25 show an example of a series of binding and extensionreactions with only the SPE's fixed to an array. In this example, SPE-P1is a primer fixed to Locus P that is complementary to the (+) strand oftarget P and P2 is a primer that is free in solution and iscomplementary to the (−) strand of target P. SPE-Q1 is a primer fixed toLocus Q that is complementary to the (+) strand of target Q and Q2 is aprimer that is free in solution and is complementary to the (−) strandof target Q.

It can be seen in FIGS. 22-25 that the specificity of the reaction andanchoring of the amplfication to a specific locus can be entirelydirected by this 1^(st) strand copying reaction. As such, the identityof the primers that are free in solution are not important as long asthey are capable of synthesizing nucleic acids that can specificallybind to the SPE's on the array. Thus although, unique specific sequenceswere used in FIGS. 22-25 for illustration of 2^(nd) strandpriming/extension reactions, in this aspect of the invention where amixed phase amplification is carried out, the primers for synthesis of2^(nd) strands could also be a carried out by a mixture of UPE's or theycan even comprise a pool of or random primers. This particular aspect ofthe present invention also finds use with general arrays that representmultitudes of variations of sequences. For instance, an array that iscreated by in situ synthesis as described by Affymatrix can besynthesized with some or all of the 65,536 permutations of an octamerarray and then used in conjunction with UPE's in solution.

Another aspect of the present invention discloses novel methods,compositions and kits for the preparation and use of protein and ligandarrays which serve to increase the exposure of the binding substance onthe array and decrease non-specific binding to the matrix itself. In oneembodiment, chimeric compositions are disclosed that are comprised oftwo segments, a nucleic acid portion and a non-nucleic portion. Thenucleic acid portion is used to achieve a practical and more accessiblemethod for attaching the non-nucleic acid portion to a solid support. Inone method of use, the nucleic acid portion is directly bound to thesurface of the array where it serves as a linker between the arraysurface and the non-nucleic acid portions of the chimeric compositions.In addition, due to the phosphate charges of the nucleic acid, eachchimeric composition at a locus should exhibit repulsive forces thatshould minimize interactions between the chimeric compositions.

Since use is being made of its physical properties rather than itssequence identity, any particular sequence can be used generically forall the various chimeric compositions. Information on the identity ofthe non-nucleic acid portion is not derived from the nucleic acidportion but rather form the spatial location on the array where thechimeric composition has been fixed or immobilized. This is in contrastto prior art, which intrinsically required a diversity of specificsequences for the nucleic acid portion and a subsequent “decoding” ofthe nucleic acid portion. In another embodiment of the presentinvention, the nucleic acid portion of the chimeric compositioncomprises discrete sequences that allow binding of the chimericcomposition to the array through hybridization to complementarysequences that are immobilized on the support.

The nucleic acid portion of a chimeric composition can be comprised ofdeoxynucleotides, ribonucleotides, modified nucleotides, nucleic acidanalogues such as peptide nucleic acids (PNAs), or any combinationthereof. The sequence of the nucleic acid portion is of completelyarbitrary nature and may be chosen by the user. In one aspect of thepresent invention, advantage is taken of the intrinsic properties ofnucleic acid hybridization for the attachment of the non-nucleic acidportion to the solid surface used for the array. Thus, the presentinvention allows the high specificity, tight binding and favorablekinetics that are characteristic of nucleic acid interactions to beconveyed to a non-nucleic acid portion that does not enjoy theseproperties.

The non-nucleic acid portion of the chimeric composition of the presentinvention can be comprised of peptides, proteins, ligands or any othercompounds capable of binding or interacting with a corresponding bindingpartner. Peptides and proteins can be comprised of amino acid sequencesranging in length from small peptides to large proteins. This peptidesand proteins can also comprise modified amino acids or analogues ofamino acids. The amino acids or analogues can comprise any desirablesequence. For instance, the amino acid sequences can be derived fromenzymes, antibodies, antigens, epitopes of antigens, receptors andglycoproteins. When peptides or proteins are used as the non-nucleicacid portion of the chimeric composition, the sequences of the nucleicacid portion are of arbitrary nature and have no correspondence to theamino acid sequences of the peptides or proteins. Other moleculesbesides peptides and proteins may also find use in the presentinvention. Examples of other constituents that could be used for thenon-nucleic acid portion can comprise but not be limited to ligands ofMW of 2000 or less, substrates, hormones, drugs and any possible proteinbinding entity.

As described previously, the particular sequence of the nucleic acid isdetermined by the user. In one method of use of the present invention,each individual species that is used as the non-nucleic acid portion canbe covalently joined to a unique nucleic acid sequence. Hybridization ofa the nucleic acid portion of the chimeric composition to acomplementary sequence at a particular locus on an array therebydetermines the identity of the particular species of the non-nucleicacid portion that is now bound to that locus. For example, one hundreddifferent chimeric compositions can be synthesized that each comprises aunique peptide and a unique nucleic acid sequence. Hybridization canthen be carried out with an array that has one hundred different loci,where each locus has nucleic acids complementary to one of the uniquenucleic acid sequences. Hybridization thereby results in thelocalization of each unique peptide to one particular locus on thearray, transforming a nucleic acid array into a peptide array. A usefulmethod for selection of sequences that could be used for the nucleicacid portion has been described by Hirschhorn et al., (op.cit.) herebyincorporated by reference. Also, since no relationship is requiredbetween the non-nucleic portion and the nucleic acid portion, adifferent set of one hundred chimeric compositions can be designed thathave different species used for the non-nucleic acid portion but use thesame set of one hundred sequences for the nucleic acid portion. In thisway, a generic nucleic acid array can be used to create differentpeptide arrays by changing the identities of the chimeric compositions.

Alternatively, non-nucleic acid protein binding substances can beattached to oligonucleotides which all comprise the same sequence. Forexample, chimeric compositions with various non-nucleic portions couldbe synthesized where the nucleic acid portion of each chimericcompositions comprised a common poly T sequence. The matrix can beprepared so that the oligonucleotides at each site consist ofcomplementary Poly A sequences. The chimeric compositions can then beapplied to the matrix using an addressable arraying system that has beendescribed by Heller et al. in U.S. Pat. No. 5,605,662 (hereinincorporated by reference). By these means, each particular chimericcomposition can be applied individually to the matrix using anelectronically controlled system and immobilized through hybridizationto the appropriate site.

The chimeric compositions at a particular locus of an array do not haveto be completely uniform in nature, i.e. an oligonucleotide sequence canbe attached to several different species of non-nucleic acid portions.For example, a series of one hundred peptides can be placed on the arrayin only four different sites by making Pool 1 with twenty-five peptidesconjugated to oligonucleotide 1, Pool 2 with twenty-five peptidesconjugated to oligonucleotide 2, Pool 3 with twenty-five peptidesconjugated to oligonucleotide 3 and Pool 4 with twenty-five peptidesconjugated to oligonucleotide 4. Attachment of the various pools ofchimeric compositions to each locus can be carried out by havingoligounucleotide 1, 2, 3 and 4 comprising unique sequences complementaryto different oligonucleotides at each site or as described above, anaddressable arraying system can be used to localize each pool usingnucleic acid portions with identical sequences. The chimericcompositions comprised of nucleic acid and non-nucleic acid portions canbe synthesized using any method known to those skilled in the art.Methods that may find use with the present invention are described in areview by Tung, C.-H.; 2000 Bioconjugate Chemistry 11, 5, 605-618) andEngelhardt et al., U.S. Pat. No. 5,241,060, issued Aug. 31, 1993 andPergolizzi et al., U.S. patent application Ser. No. 08/479,995, filedJun. 7, 1995, for Analyte Detection Utilizing Polynucleotide Sequences,Composition, Process and Kit, based on priority U.S. patent applicationSer. No. 06/491,929, filed May 5, 1983, all incorporated herein byreference. In one approach, peptides and oligonucleotides aresynthesized separately using standard automated procedures and thencovalently bonded together. For example, a thiol group can be addedeither to the 5′-terminus of the oligonucleotide or internally in thenucleic acid portion of the chimeric composition. Addition of amaleimido group to the N-terminus or in an internal position of thepeptide allows a reaction with the thiol group of the oligonucleotide toform a chimeric composition comprised of a nucleic acid and a peptide(Eritja et al., (1991) Tetrahedron, 47; 4113-4120. Arar et al.; (1993)Tetrahedron Lett 34; 8087-8090, Ede et al., (1994) BioconjugateChemistry 5; 373-378, Stetsenko and Gait, (2000) J. Org. Chem. 65;4900-4908). Alternatively the chimeric composition can be prepared bythe stepwise addition of amino acids and nucleotides on the same solidsupport, (de la Torre et al., (1994) Tetrahedron Lett 35; 2733-2736,Bergmann and Bannwarth (1995) Tetrahedron Lett. 36; 1839-1842, Robles etal., (1999) Tetrahedron 55; 13,251-13,264, Antopolsky et al., (1999)Helv. Chim Acta 82; 2130-2140). In these publications each of which isincorporated by reference herein, the peptide was synthesized firstfollowed by the addition of bases to synthesize the oligonucleotideportion. In standard peptide synthesis, the N-terminus and the sidechains of the amino acids are protected by Fmoc and tert-butyl groupsrespectively. At each cycle the Fmoc group is removed with 20%piperidine and the side chains are deprotected with 90% trifluoroaceticacid. However when both oligonucleotides and peptides were synthesizedas part of a single composition, different chemistries had to be used.For example, base labile Fmoc and 9-fluorenylmethyl groups were used asthe amino acid side chain protecting groups to avoid exposing the DNA tostrong acids (de la Torre, op cit.; de la Torre et. al., 1999Bioconjugate Chem. 10; 1005-1012; Robles et al op cit.), all suchpublications being incorporated by reference herein. Methods for makingchimeric compositions of peptides fused to PNA analogues of nucleicacids have been described by Cook et al. in U.S. Pat. No. 6,204,326,incorporated herein by reference. Furthermore, chimeric compositionscomprised of nucleic acids and peptides can be synthesized directly on asolid surface to create an array using the methods described by Sundberget al in U.S. Pat. No. 5,919,523 incorporated herein by reference.

The solid support can be any material used for arrays including, but notlimited to nylon or cellulose membranes, glass, synthetic, plastic,metal. The materials can be opaque, reflective, transparent ortranslucent. They can be porous or they can be non-porous. Nucleic acidsthat are either part of chimeric compositions or meant to becomplementary to chimeric compositions can be affixed to the solidsupport by any previously known methods used to prepare DNA arrays.

Binding of analytes to appropriate binding partners can be carried outin either a mixed phase or a liquid phase format. For instance, thepresent invention has disclosed the direct fixation of bindingsubstances to the array by the use of rigid arm linkers and chimericcompositions. The binding substance on the array (the solid phase) canbe exposed to a solution (the liquid phase) that contains the analytesof interest. Interactions between the binding substance on the array andanalytes in solution can then later be quantified. Examples of theinteractions that may find use in the present invention can comprise butnot be limited to peptide-protein, antigen-antibody, ligand-receptor orenzyme-substrates. For example, an array can be prepared with a seriesof peptides to determine their ability to bind to a particular antibody.The array is incubated in a solution containing the antibody followed bywashing away the unbound material. Detection of the antibody bound tovarious components on the array can then be carried out by any of anumber of conventional techniques. For instance, in this example theantibody that is applied to the array can be labeled with biotin forindirect detection, or a fluorescent compound for direct detection.Alternatively, the antibody analyte is unlabeled and a secondaryantibody can be utilized which either has a fluorescent label for directdetection or indirect label such as biotin. Thus, in this example theantibody-antigen interaction occurs with the antigen bound to the solidmatrix.

The present invention has also disclosed the use of chimericcompositions that are indirectly bound to the array throughhybridization of the nucleic acid portions of the chimeric compositionsto complementary nucleic acids fixed or immobilized to the array. Thesecan be used in the in the same mixed phase format that has beendescribed above by hybridization of the chimeric compositions to thearray followed by binding of analytes. However, the use of hybridizationto immobilize the chimeric compositions to specific loci on the arrayallows the use of a completely liquid phase format for binding ofanalytes to the chimeric compositions. In this way, the chimericcompositions can be combined with the target molecules in solution underoptimal conditions for interactions between the analyte and thenon-nucleic acid portions of the chimeric compositions. The resultantsolution, containing the chimeric compositions free in solution as wellas the chimeric compositions that are bound into complexes with theanalytes, can then be applied to the matrix and the various chimericcompositions will be localized to various locations on the array throughhybridization to the nucleic acid portion to complementary sequences onthe array. An illustration of this process is given in FIG. 28.

The hybridization can be carried out under mild conditions, which willnot interfere with the ligand-receptor or protein-protein complex.Protein-protein interactions are generally characterized by low Km's, inthe order of magnitude of 10⁻⁵ to 10⁻⁹. In this aspect of the presentinvention, the protein interactions can occur in solution rather than ona solid surfaces which will allow superior kinetics of binding and willalso allow a wider variety of conditions for protein binding than can beobtained in the mixed format. Also, by chimeric compositions andanalytes together in solution, direct interaction or interference withthe matrix is avoided, thereby decreasing the background. Therefore, touse the example cited before, the solution containing the antibodytarget is combined with a solution containing the chimeric composition.Thus, by using the methods of the present invention, the proteins willremain in solution throughout the process preventing any problemsassociated with dehydrating the protein bound to the solid matrix.

The method of the present invention can be used to study many systemsthat involve interactions between compound. These can include but not belimited to antigen-antibody relationships, protein-protein interactions,enzyme-substrate receptor-ligand interactions, ligand-receptor,hormone-receptor, carbohydrate-lectins, drug screening, and patterns ofexpression of proteins in a cell or tissue. Another method of use of thepresent invention is that instead of using unique nucleic acid portionsfor each individual non-nucleic acid portion, one specific bindingsubstance can be combined with various nucleic acid sources to form agroup of chimeric compositions with a common non-nucleic acid portionand a unique nucleic acid portion. Each particular chimeric compositioncan be combined with an analyte from a different source and applied tothe array by hybridizing the nucleic acid portions to theircomplementary sequences on the array. The proteins bound to the arraycan then be detected following standard procedures. By these means, theamount of targets from each source that can interact with the bindingsubstance in the chimeric compositions can be simultaneously determined.

For instance, a set of twenty different compositions can be synthesizedwhere each member of the set will have a different nucleic acid portionbut the same peptide. Another set can be made with a different peptidethat is linked to twenty other nucleic acid portions. More sets can bemade on the same basis. Protein extracts can then be made from twentydifferent tissues and each extract can be combined with a differentmember of the set of chimeric compositions. Thus, the nucleic acidportion serves as a marker for not only the peptide but also for theparticular tissue that was used as the source. For instance, a group ofsets can be made with peptides that have affinities for differentreceptors. After incubation of the mixtures with the chimeric compounds,the mixtures are applied to the array and detected. In this way, eachparticular receptor that is being studied can be quantified and comparedsimultaneously between various tissues. Alternatively, the same nucleicacid sequence can be used in common for each source by using theaddressable system described previously, and carrying out hybridizationto each locus after addition of each individual reaction mixture.

The same method can be applied to tissues or cell cultures that are fromthe same source but are treated differently. For example, in a drugdiscovery program, nine different drugs can be added to individual cellcultures to determine the effect on specific proteins. Chimericcompositions are designed and synthesized with peptides that are knownto react with each of proteins that is to be monitored. As in theprevious example, a specific nucleic acid sequence will serve as amarker for each peptide and each particular treatment. The proteins areextracted from each of the ten cell cultures (nine drug treated plus anuntreated control) and incubated with the chimeric compositions. Themixtures are applied to the array and the amount of analyte bound to thecorresponding peptides at each locus of the array is measured for thevarious drug conditions. If desired, the present invention can also beused for the isolation of analytes. This can be carried out by eitherdisrupting the interaction between the analyte and the non-nucleic acidportion of the chimeric compositions or by denaturing the nucleic acidportion from the complementary sequence fixed or immobilized to thearray. It is also contemplated that removal of chimeric compositionsfrom the array may also allow the reuse of the array in otherexperiments.

In further detail, this invention provides novel chimeric compositionsand processes using such chimeric compositions. One such composition ofmatter comprises an array of solid surfaces comprising a plurality ofdiscrete areas, wherein at least two of such discrete areas comprise: achimeric composition comprising a nucleic acid portion; and anon-nucleic acid portion, wherein the nucleic acid portion of a firstdiscrete area has the same sequence as the nucleic acid portion of asecond discrete area, and wherein the non-nucleic acid portion has abinding affinity for analytes of interest.

Another composition of matter comprises an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of thediscrete areas comprise a chimeric composition hybridized tocomplementary sequences of nucleic acids fixed or immobilized to thediscrete areas, wherein the chimeric composition comprises a nucleicacid portion, and a non-nucleic acid portion, the nucleic acid portioncomprising at least one sequence, wherein the non-nucleic acid portionhas a binding affinity for analytes of interest, and wherein when thenon-nucleic acid portion is a peptide or protein, the nucleic acidportion does not comprises sequences which are either identical orcomplementary to sequences that code for such peptide or protein.

Mention should be made of a process for detecting or quantifyinganalytes of interest, the process comprising the steps of 1) providinga) an array of solid surfaces comprising a plurality of discrete areas,wherein at least two of such discrete areas comprise a chimericcomposition comprising a nucleic acid portion, and a non-nucleic acidportion; wherein the nucleic acid portion of a first discrete area hasthe same sequence as the nucleic acid portion of a second discrete area;and wherein the non-nucleic acid portion has a binding affinity foranalytes of interest; b) a sample containing or suspected of containingone or more of the analytes of interest; and c) signal generating means;2) contacting the array a) with the sample b) under conditionspermissive of binding the analytes to the non-nucleic acid portion; 3)contacting the bound analytes with the signal generating means; and 4)detecting or quantifying the presence of the analytes.

Another process for detecting or quantifying analytes of interestcomprises the steps of 1) providing a) an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of suchdiscrete areas comprise a chimeric composition comprising a nucleic acidportion; and a non-nucleic acid portion; wherein the nucleic acidportion of a first discrete area has the same sequence as the nucleicacid portion of a second discrete area; and wherein the non-nucleic acidportion has a binding affinity for analytes of interest; b) a samplecontaining or suspected of containing one or more of the analytes ofinterest; and c) signal generating means; 2) labeling the analytes ofinterest with the signal generating means; 3) contacting the array a)with the labeled analytes under conditions permissive of binding thelabeled analytes to the non-nucleic acid portion; and 4) detecting orquantifying the presence of the analytes.

Another process for detecting or quantifying analytes of interestcomprises the steps of 1) providing a) an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of suchdiscrete areas comprise nucleic acids fixed or immobilized to suchdiscrete areas, b) chimeric compositions comprising: i) a nucleic acidportion; and ii) a non-nucleic acid portion; the nucleic acid portioncomprising at least one sequence, wherein the non-nucleic acid portionhas a binding affinity for analytes of interest, and wherein when thenon-nucleic acid portion is a peptide or protein, the nucleic acidportion does not comprise sequences which are either identical orcomplementary to sequences that code for the peptide or protein; c) asample containing or suspected of containing the analytes of interest;and d) signal generating means; 2) contacting the array with thechimeric compositions to hybridize the nucleic acid portions of thechimeric compositions to complementary nucleic acids fixed orimmobilized to the array; 3) contacting the array a) with the sample b)under conditions permissive of binding the analytes to the non-nucleicacid portion; 4) contacting the bound analytes with the signalgenerating means; and 5) detecting or quantifying the presence of theanalytes.

Another process for detecting or quantifying analytes of interestcomprises the steps of 1) providing a) an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of thediscrete areas comprise nucleic acids fixed or immobilized to thediscrete areas, b) chimeric compositions comprising i) a nucleic acidportion; and ii) a non-nucleic acid portion, the nucleic acid portioncomprising at least one sequence, wherein the non-nucleic acid portionhas a binding affinity for analytes of interest, and wherein when thenon-nucleic acid portion is a peptide or protein, the nucleic acidportion does not comprise sequences which are either identical orcomplementary to sequences that code for the peptide or protein; c) asample containing or suspected of containing the analytes of interest;and d) signal generating means; 2) contacting the chimeric compositionswith the sample b) under conditions permissive of binding the analytesto the non-nucleic acid portion; 3) contacting the array with thechimeric compositions to hybridize the nucleic acid portions of thechimeric compositions to complementary nucleic acids fixed orimmobilized to the array; 4) contacting the bound analytes with thesignal generating means; and 5) detecting or quantifying the presence ofthe analytes.

Another useful process comprises the steps of 1) providing a) an arrayof solid surfaces comprising a plurality of discrete areas; wherein atleast two of the discrete areas comprise nucleic acids fixed orimmobilized to the discrete areas, b) chimeric compositions comprisingi) a nucleic acid portion; and ii) a non-nucleic acid portion; thenucleic acid portion comprising at least one sequence, wherein thenon-nucleic acid portion has a binding affinity for analytes ofinterest, and wherein when the non-nucleic acid portion is a peptide orprotein, the nucleic acid portion does not comprise sequences which areeither identical or complementary to sequences that code for the peptideor protein; c) a sample containing or suspected of containing theanalytes of interest; and d) signal generating means; 2) contacting thearray with the chimeric compositions to hybridize the nucleic acidportions of the chimeric compositions to complementary nucleic acidsfixed or immobilized to the array; 3) labeling the analytes of interestwith the signal generating means; 4) contacting the array with thelabeled analytes to bind the analytes to the non-nucleic acid portion;and 5) detecting or quantifying the presence of the analytes.

Another process for detecting or quantifying analytes of interestcomprises the steps of 1) providing a) an array of solid surfacescomprising a plurality of discrete areas; wherein at least two of thediscrete areas comprise nucleic acids fixed or immobilized to thediscrete areas, b) chimeric compositions comprising: i) a nucleic acidportion; and ii) a non-nucleic acid portion; the nucleic acid portioncomprising at least one sequence, wherein the non-nucleic acid portionhas a binding affinity for analytes of interest, and wherein when thenon-nucleic acid portion is a peptide or protein, such nucleic acidportion does not comprise sequences which are either identical orcomplementary to sequences that code for the peptide or protein; c) asample containing or suspected of containing the analytes of interest;and d) signal generating means; 2) contacting the array with thechimeric compositions to hybridize the nucleic acid portions of thechimeric compositions to complementary nucleic acids fixed orimmobilized to the array; 3) labeling the analytes of interest with thesignal generating means; 4) contacting the array with the labeledanalytes to bind the analytes to the non-nucleic acid portion; and 5)detecting or quantifying the presence of the analytes.

The elements recited in the last several chimeric compositions andprocesses using such chimeric compositions are described elsewhere inthis disclosure.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Amplification of aLibrary of RNA Targets with 2^(nd) Strand Synthesis Carried Out byRandom Primers with T7 Promoter Sequences

1) First Strand Synthesis

Two mixtures of 250 ng of rabbit globulin mRNA (Life Technologies,Rockville, Md.) and 200 ng of Oligo (dT)₂₄ (In house or purchased?) in 5ul were heated at 70° C. for 10 minutes followed by a 2 minuteincubation on ice. This material was then used in 10 ul reactionscontaining 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT,600 uM dNTPs and 120 units of Superscript II RNase H ReverseTranscriptase (Life Technologies, Rockville, Md.) with incubation at 42°C. for 60 minutes.

2) Second Strand Synthesis

KOH was added to the reactions for a final concentratiion of 200 mM.Incubation was carried out at 37° C. for 30 minutes followed byneutralization with an equimolar amount of glacial acetic acid. Primerswith the following sequence were used for 2^(nd) strand synthesis:5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGN₁₋₉-3′Primers with the sequence above (TPR primers) consist of a T7 promotersequence at their 5′ ends and 9 nucleotides with random sequences attheir 3′ ends. 400 pmoles of TPR primers and other appropriate reagentswere added for a final reaction mix of 30 ul containing 86.6 mM Tris-HCl(pH 7.6), 32 mM KCl, 200 mM KOAc (??), 15.6 mM MgCl₂, 3.3 mM DTT, 10 mMDithioerythritol (DTE), 10 mM (NH₄)₂SO₄, 0.15 mM-NAD, 200 ug/mlnuclease-free BSA (Bayer, Kankakee, Ill.), Annealing was carried out byheating the mixture to 65° C. and slow cooling to room temperaturefollowed by incubation on ice for 5 minutes. Extension of the primerswas carried out by addition of 1.2 ul of 10 mM dNTPs, 4 units of E. coliDNA ligase (New England Biolabs, Beverly, Mass.) and either 12 units ofDNA polymerase I (New England Biolabs, Beverly, Mass.) or 6 units of theExo (−) version of the Klenow fragment of DNA Polymerase I (New EnglandBiolabs, Beverly, Mass.). Incubation was carried out at 15° C. for 5minutes followed by 37° C. for 120 minutes. The reactions were puriifedby extraction with Phenol/Chloroform with Phase-Lock Gels (Eppendorf,Westbury, N.Y.) and Ethanol precipitated.3) Transcription

Transcription was carried out by using the BioArray High YieldTranscription Kit (T7) (ENZO Diagnostics, Farmingdale, N.Y.) followingthe manufacturers instructions with a final volume of 40 ul. Thereaction mixes also contained 10 uCi of ³H-ATP with a specific activityof 45 Ci/mMol (Amersham Pharmacia, Piscataway, N.J.). Incorporation wasmeasured by addition of 5 ul of the transcription reaction to 1 ml of10% TCA, 50 ug/ml Poly A, 5 mM EDTA followed by incubation on ice for 30minutes. Precipitates were collected on 25 mm glass fiber filters(Whatman, Lifton, N.J.) followed by three washes with 5% TCA and threewashes with ethanol.

4) Results and Conclusions

-   Sample 1 with DNA polymerase I 4,243 cpm-   Sample 2 with Exo (−) Klenow 19,662 cpm    This example demonstrated that RNA transcripts were obtained from a    library of nucleic acids by the steps described above and that under    the conditions used, the Exo (−) version of Klenow resulted in more    product compared to the use of DNA polymerase I.

EXAMPLE 2 Amplification of a Library of RNA Targets with 1^(st) StrandSynthesis Using Oligo-T Magnetic Beads and 2^(nd) Strand SynthesisCarried Out by Random Primers with T7 Promoter Sequences

1) Preparation of Beads

50 ul of Dynal Oligo (dT)₂₅ magnetic beads (Dynal Inc., Lake Success,N.Y.) were washed two times with 100 ul of Binding Buffer (20 mMTris-HCl (pH 7.5), 1.0 M LiCl, 2 mM EDTA) and then resuspended in 50 ulof Binding Buffer.

2) Binding of RNA to Beads

RNA targets were prepared by diluting 1 ug of mouse poly A RNA (SigmaChemical Co, St. Louis, Mo.) or 1 ug of wheat germ tRNA (Sigma ChemicalCo, St. Louis, Mo.) into RNase-free H₂O (Ambion, Austin, Tex.) for afinal volume of 50 ul, and heating the RNA solution at 65° C. for 5minutes. The RNA solution was combined with the beads prepared in Step 1and mixed for 15 minutes at room temperature with a Dynal Sample Mixer(Dynal Inc., Lake Success, N.Y.). Unbound material was removed bymagnetic separation with a Dynal Magnetic Particle Concentrator (Dynal,Inc. Lake Success, N.Y.) followed by two washes with 200 ul of WashBuffer B (10 mM Tris-HCl (pH 7.5), 150 mM LiCl, 1 mM EDTA) and threewashes with 250 ul of First Strand Buffer (50 mM Tris-HCl (pH 8.3), 75mM KCl, 3 mM MgCl₂)

3) First Stand Synthesis

The beads from Step 2 were resuspended in 50 mM Tris-HCl (pH 8.3), 75 mMKCl, 3 mM MgCl₂. 10 mM DTT, 500 uM dNTPs and 400 units of Super ScriptII RNase H Reverse Transcriptase (Life Technologies, Rockville, Md.) andincubated for 90 minutes at 42° C.

4) Second Strand Synthesis

RNA templates were removed by heating the First Strand Synthesisreaction mixture of step 3 at 90° C. for 5 minutes followed by removalof the supernatant after magnetic separation. The beads were washed twotimes with 100 ul of Buffer C (70 mM Tris-HCl (ph 6.9) 90 mM KCl, 14.6mM MgCl₂,10 mM DTE, 10 mM (NH₄)₂SO4 and 200 ug/ml nuclease-free BSA) andresuspended in 50 ul of Random Priming Mix A (86.7 mM Tris-HCl (pH 7.6),113.3 mM KCl, 17 mM MgCl₂, 11.3 mM DTT, 11.3 mM (NH₄)₂SO₄, 227 ug/mlnuclease-free BSA) containing 360 pmoles of TPR primers. Primers wereallowed to anneal on ice for 15 minutes. Unbound primers were removed bymagnetic separation. The beads were resuspended in 50 ul of RandomPriming Mix A (without the TPR primers) with 10 units of the Klenowfragment of DNA Polymerase I (New England. Biolabs, Beverly, Mass.) and400 mM dNTP's. Incubation was carried out for 5 minutes at 4° C., 30minutes at 15° C., and 30 minutes at 37° C. For some samples, anadditional 25 ul of Oligo T magnetic beads prepared as described in Step1 were washed with Buffer C and added to the reaction mix. Also, forsome samples, 3 units of T4 DNA Polymerase (New England Biolabs,Beverly, Mass.) and 2 ul of a 10 mM stock of dNTPs were added to thereaction mixtures. Samples with these further steps were incubated for30 minutes at 37° C. At the conclusion of the varied reactions, thebeads were magnetically separated from the reagents. and the beads wereused to carry out transcription assays.

5) Transcription Synthesis

Transcription reactions were carried out by resuspending the beads inreagents from the BioArray High Yield Transcription Kit (T7) (ENZODiagnostics, Farmingdale, N.Y.) using the manufacturer's instructionswith a final volume of 40 ul. The reaction mixtures also contained 10uCi of ³H-ATP with a specific activity of 45 Ci/mMol (AmershamPharmacia, Piscataway, N.J.). Extent of transcription was measured byusing TCA precipitation as described previously.

6) Results Extra T4 DNA Sample Target Beads polymerase cpm Incorporated1 Poly A (−) (−) 8,535 2 Poly A (−) (+) 15,483 3 Poly A (+) (−) 16,048 4Poly A (+) (+) 18,875 5 tRNA (+) (+) 2,5487) Conclusions

This example demonstrated that transcripts were obtained from a libraryof nucleic acids by the steps described above. Addition of extra beadscan increase the amount of synthesis. The reaction can be carried outwithout a T4 DNA polymerization step but the amount of synthesis can beincreased by the addition of such a reagent.

EXAMPLE 3 Dependency on Reverse Transcriptase for Amplification of aLibrary of RNA Targets with Oligo-T Magnetic Beads and Random Primerswith T7 Promoter Sequences

1) Preparation of Beads

This step was carried out as described in Step 1 of Example 2, exceptthe amount of beads was increased to 100 ul for each reaction.

2) Binding of RNA to Beads

RNA targets were prepared by diluting 1 ug of mouse poly A mRNA (SigmaChemical Co, St. Louis, Mo.) into nuclease-free H₂O (Ambion Inc.,Auistin Tex.) for a final volume of 50 ul, and heating the RNA solutionat 65° C. for 15 minutes. The RNA solution was combined with the beadsprepared in Step 1 and mixed for 15 minutes at Room Temperature with aDynal Sample Mixer (Dynal Inc., Lake Success, N.Y.). Unbound materialwas removed by magnetic separation. followed by two washes with 200 ulof Wash Buffer B and two washes with 100 ul of First Strand Buffer.

3) First Strand Synthesis

This step was carried out as describe d in step 3 of Example 2 exceptthat a pair of duplicate samples had the Reverse Transcriptase omitted.

4) Second Strand Synthesis

RNA templates were removed by heating the First Strand Synthesisreaction mixture of step 3 at 90° C. for 4 minutes followed by removalof the supernatant after magnetic separation. The beads were washed twotimes with 100 ul of Wash Buffer B and resuspended in 50 ul of RandomPriming Mix A containing 360 pmoles of TPR primers. Primers were allowedto anneal on ice for 15 minutes. Unbound primers were removed bymagnetic separation and the beads were washed twice with 100 ul of coldBuffer D (20 mM Tris-HCl (pH 6.9), 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂SO₄. The beads were then suspended in 40 ul of Buffer C that alsocontained 1 mM dNTPs and 10 units of the Klenow fragment of DNAPolymerase I (New England Biolabs, Beverly, Mass.). Incubation wascarried out for 5 minutes at 4° C., 30 minutes at 15° C., and 30 minutesat 37° C. The reaction was carried out further by the addition of 2 ul(6 units) of T4 DNA Polymerase (New England Biolabs, Beverly, Mass.) and2 ul of a 10 mM stock of dNTPs, followed by incubation for 30 minutes at37° C.

5) Transcription Synthesis

The beads were washed two times with 100 ul of Wash Buffer B and oncewith 100 ul of 10 mM Tris-HCl (pH 7.5). The beads were resuspended in 10ul of 10 mM Tris-HCl (pH 7.5) and mixed with reagents from a BioArrayHigh Yield Transcription Kit (T7) (ENZO Diagnostics, Farmingdale, N.Y.)using the manufacturer's instructions. The volume of the reaction was 30ul and the incubation was carried out for 2 hours at 37° C.

6) Results and Conclusions

Analysis of the reaction was carried out by gel electrophoresis of 10 ulof the transcription reaction using 1% Agarose in 0.5× TBE buffer. Theresults of this experiment are in FIG. 27 for duplicate samples anddemonstrate that transcripts were obtained from a library of nucleicacids by the steps described above and this synthesis was dependent uponthe presence of Reverse Transcriptase activity.

EXAMPLE 4 Multiple Rounds of Synthesis of 2^(nd) Strands by RandomPrimers with T7 Promoters

Steps 1, 2 and 3 for Preparation of beads, binding of mRNA and 1 Ststrand synthesis were carried out as described in steps 1 through 3 ofExample 3.

4) Second Strand Synthesis

After 1^(st) strand synthesis, the liquid phase was removed by magneticseparation and the beads resuspended in 100 ul of Detergent Wash No.1(10 mM Tris-HCl (pH 7.5), 1% SDS) and heated at 90° C. for 5 minutes.The supernatant was removed by magnetic separation and the beads werewashed with 100 ul of Detergent Wash No.2 (40 mM Tris-HCl (pH 8.0), 200mM KCl, 0.2 mM EDTA, 0.01% Tween 20, 0.01% Nonidet P40). The beads werewashed two times with 100 ul of Wash Buffer B and resuspended in 50 ulof Random Priming Mix A containing 360 pmoles of TPR primers. Primerswere allowed to anneal on ice for 15 minutes. Unbound primers wereremoved by magnetic separation and the beads were washed twice with 100ul of cold Buffer D (20 mM Tris-HCl (pH 6.9), 90 mM KCl, 4.6 mM MgCl₂,10 mM DTT, 10 mM (NH₄)₂SO4). The beads were then suspended in 40 ul ofBuffer C that also contained 1 mM dNTPs and 10 units of the Klenowfragment of DNA Polymerase I (New England Biolabs, Beverly, Mass.).Incubation was carried out for 5 minutes at 4° C., 30 minutes at 15° C.,and 30 minutes at 37° C. The reaction was carried out further by theaddition of 2 ul (6 units) of T4 DNA Polymerase (New England Biolabs,Beverly, Mass.) and 2 ul of a 10 mM stock of dNTPs, followed byincubation for 30 minutes at 37° C. The beads were then washed two timeswith 100 ul of Wash Buffer B, resuspended in 50 ul of 10 mM Tris-HCl (pH7.5) and heated at 90° C. for 5 minutes. The supernatant was removedafter magnetic separation and store as supernatant No. 1. The beads werethen washed once with 100 ul of Detergent Wash No.2, two times with 100ul of Wash Buffer B and resuspended in 50 ul of Random Priming Mix Acontaining 360 pmoles of TPR primers. Primer annealing and extension wascarried out as described above. The beads were then washed two timeswith 100 ul of Wash Buffer B, resuspended in 50 ul of 10 mM Tris-HCl (pH7.5) and heated at 90° C. for 5 minutes. The supernatant was removedafter magnetic separation and store as supernatant No.2. The series ofwashes, annealing and extension steps were carried out again using thesteps described above. The beads were then washed two times with 100 ulof Wash Buffer B, resuspended in 50 ul of 10 mM Tris-HCl (pH 7.5) andheated at for 5 minutes. The supernatant was removed after magneticseparation and stored as supernatant No.3.

5) Synthesis of Complements to the 2^(nd) Strands

A pool was created by combining supernatant No.1, supernatant No.2 andsupernatant No.3. This pool comprises a library of 2^(nd) strands freein solution with T7 promoters at their 5′ ends and poly A segments attheir 3′ ends. Fresh magnetic beads with poly T tails were prepared andannealed to the pool of 2^(nd) strands by the same processes describedin Steps 1 and 2 of Example 2. Extension was then carried out byresuspension of beads in 50 ul of Buffer C that also contained 1 mMdNTPs and 10 units of the Klenow fragment of DNA Polymerase I (NewEngland Biolabs, Beverly, Mass.). Incubation was carried out at 37° C.for 90 minutes. Transcription was then carried out as described in step5 of Example 3 except the reaction volume was reduced to 20 ul.

6) Results and Conclusions

The results of this experiment are in FIG. 28 and demonstrated thattranscripts were obtained from a library of polyA mRNA by the stepsdescribed above. This example demonstrated that a library of 2^(nd)strands was obtained after multiple rounds of 2^(nd) strand synthesis,isolated free in solution and then used to create functionally activeproduction centers.

EXAMPLE 5 Additional RNA Synthesis from Transcription Constructs

The library of transcription constructs described in Example 4 were usedfor a second round of transcription. After removal of transcriptionproducts for analysis in Example 4, the beads were resuspended in 100 ulof 10 mM Tris-HCl (pH 7.5) and left overnight at 4° C. The next day, thebeads were washed with 100 ul of Detergent Wash No.2, resuspended in 100ul of Detergent Wash No.1 and heated at 42° C. for 5 minutes followed bytwo washes with 100 ul of Detergent Buffer No.2, two washes with 100 ulof Wash Buffer B and two washes with 100 ul of 10 mM Tris-HCl (pH 7.5).A transcription reaction was set up as described previously with a 20 ulvolume.

Results and Conclusions

Results of the transcription reaction are shown in FIG. 29 and show thatthe nucleic acids synthesized in Example 4 were stable and could be usedfor additional transcription synthesis.

EXAMPLE 6 Terminal Transferase Addition of Poly G Tail to 1^(st) Strandsfor Binding of Primers with T7 Promoter

1) Preparation of Beads

150 ul of Dynal Oligo (dT)₂₅ magnetic beads (Dynal Inc., Lake Success,N.Y.) were washed two times with 150 ul of Binding Buffer andresuspended in 75 ul of Binding Buffer.

2) Binding of RNA to Beads

RNA targets were prepared by diluting 3 ul of 0.5 ug/ul mouse poly A RNA(Sigma Chemical Co, St. Louis, Mo.) with 32 ul of RNase-free H₂O(Ambion, Austin, Tex.) and 40 ul of Binding Buffer, and heating the RNAsolution at 65° C. for 5 minutes. The RNA solution was combined with thebeads prepared in Step 1 and mixed for 30 minutes at room temperature.

3) First Strand Synthesis

Unbound material was removed by magnetic separation. followed by twowashes with 200 ul of Wash Buffer B and one wash with 100 ul of FirstStrand Buffer. The beads were resuspended in a 50 ul mixture of 50 mMTris-HCl (pH 7.5), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 500 uM dNTPs and400 units of Super Script II RNase H Reverse Transcriptase (LifeTechnologies, Rockville, Md.) and incubated for 90 minutes at 42° C. Atthe end of the 1^(st) strand synthesis reaction, the liquid phase wasremoved by magnetic separation and the beads resuspended in 100 ul ofDetergent Wash No.1 and heated at 90° C. for 5 minutes. The supernatantwas removed by magnetic separation and the beads were washed with 100 ulof Detergent Wash No.2, two times with 100 ul of Wash Buffer B andresuspended in 300 ul of 10 mM Tris-HCl (pH 7.5).

4) Second Strand Synthesis

Two methods were used for carrying out second strand synthesis. Thefirst method was as described for the previous examples, I.e the use ofTPR primers that have a T7 promoter on their 5′ ends and randomsequences at their 3′ ends. The second method was the use of T7-C9primers that have a T7 promoter at their 5′ ends and a poly C segment attheir 3′ ends. The sequence of the T7-C9 primers is as follows: 5′GGCCAGTGAATTGTAATACGACTCACTATAGGGATCCCCCCCCC-3′

The product of Step 3 was divided into two portions. The first portion(Sample No.1) consisted of 100 ul and was set aside to be used forrandom priming. The second portion (the remaining 200 ul) was processedfurther by magnetically separating the buffer from the beads andresuspending the beads in 100 ul and adding 100 ul of Poly A Mix (1.6ug/ul Poly A, 10 mM Tris-HCL (pH 7.5), 0.5 M LiCl, 1 mM EDTA). The PolyA was obtained from (Amersham Pharmacia, Piscataway, N.J.) and had anaverage length of 350 nucleotides. The beads and Poly A were mixedtogether for 30 minutes at room temperature with a Dynal Sample Mixer(Dynal Inc., Lake Success, N.Y.). The beads were washed two times withWash Buffer B and resuspended in 200 ul of 10 mM Tris-HCl (pH 7.5). Thiswas divided into two 100 ul portions, Sample No.2 and Sample No.3.Sample No.3 was processed further by magnetically separating the bufferfrom the beads and resuspending the beads in an 80 ul reaction mixtureusing reagents and directions from the 3′ Oligonucleotide Tailing System(ENZO Biochem, Farmingdale, N.Y. 11561) with 0.5 mM dGTP present. SampleNo.3 was incubated for one hour at 37° C. followed by removal of thereagents by magnetic separation. The beads were then resuspended in 100ul of Detergent Buffer No.1 and heated at 90° C. for 3 minutes. Thebeads were then washed once with 100 ul of Detergent Wash No.2 and twicewith 100 ul of Wash Buffer B. Sample No. 3 was resuspended in 100 ul of10 mM Tris-HCl (pH 7.5). All three samples (Sample No.1, Sample No.2 andSample No.3) were washed once with 100 ul Wash Buffer E (100 mM Tris-HClpH7.4) 20 mM KCl, 10 mM MgCl₂, 300 mM (NH₄)₂SO₄) and then resuspended in50 ul of Buffer E. Primers for 2^(nd) strand synthesis were added toeach sample: 4 ul of 100 pMole/ul of TPR primers to Sample No.1 and 4 ulof 10 pMole/ul of T7-C9 primers to Samples No.2 and No.3. Samples werethen incubated on ice for 15 minutes followed by one wash with 100 ul ofice cold Buffer E and one wash with ice cold Buffer D. Each sample wasresuspended in 40 ul of Buffer D that also contained 1 mM dNTPs and 200units of the Klenow fragment of DNA Polymerase I (New England Biolabs,Beverly, Mass.). Incubations were carried out for 30 minutes at 15° C.followed by 30 minutes at 37° C.

All three samples were further processed by the addition of 2 ul (3units) of T4 DNA polymerase (Source, Location) and 2 ul of 10 mM dNTPsfollowed by incubation at 37° C. for 30 more minutes. Samples werewashed twice with 100 ul of 10 mM Tris-HCl (pH 7.5). A Transcriptionreaction was set up as described previously with a 20 ul volume.

5) Results and Conclusions

Analysis of the reaction was carried out by gel electrophoresis with 2ul and 10 ul samples of the transcription reaction using 1% Agarose in0.5× TBE buffer. The results of this experiment are in FIG. 30 anddemonstrated that non-inherent UDTs were added to the ends of a libraryof 1^(st) strand copies by the methods described above. Thenon-inherrent UDTs served as primer binding sites for primers with polyC at their 3′ ends for synthesis of a library of 2^(nd) strands. Thedifference in the amount of RNA transcription between Samples No.2 andNo. 3 serves as a further indication that comparatively little primingtook place at internal sites under the conditions used.

EXAMPLE 7 Terminal Transferase Addition of Poly G Tail to 1^(st) Strandsfor Binding of Primers with T7 Promoter (Incorporation Assay)

The transcription products of Example 6 were analyzed by gelelectrophoresis as shown in FIG. 30. To obtain numerical evaluation ofthe method described in that example, the libraries attached to thebeads in Samples No.1, No.2 and No.3 were used in another transcriptionreaction using ³H-incorporation. Transcription was carried out asdescribed in Example 3.

The results were as follows: Random priming  6,660 cpm Sample No. 1)T7-C9 primers without TdT addition step  1,144 cpm Sample No. 2 T7-C9primers with TdT addition step 21,248 cpm Sample No. 3

This second assay agrees with the conclusions of Example 6; i.e. theT7-C9 primers can be used in the present method and more priming tookplace with the terminally added poly G sequences compared to internalsequences.

EXAMPLE 8 Incorporation of Promoters After 2^(nd) Strand Synthesis

1) Preparation of Beads

Preparation of beads for each sample was carried out as described instep 1 of Example 3

2) Binding of RNA to Beads

In each sample, 1 ug of poly mRNA was bound to beads as described instep 2 of Example 3 with the addition of having 120 units of Prime RNaseInhibitor (Eppendorf, Westbury, N.Y.) present.

3) First Strand Synthesis

First strand synthesis was carried out as described in step 3 of Example3 except the reaction was also supplemented with 120 units of PrimeRNase Inhibitor.

4) Second Strand Synthesis

Poly dG addition was carried out as described for sample No. 3 inExample 6. Second strand synthesis was performed as described in Example6 except that 80 pMoles of primers were used in 100 ul reactions. ForSamples No. 1 and No.2, the 2nd strand primers were the T7-C9 primerspreviously described. For Samples No.3 and No.4, the 2nd strand primerswere C9 primers with the sequence: 5′-CCCCCCCCC-3′. At the end of thereaction, all samples were washed twice with 100 ul 10 mM Tris-HCl (pH7.5).

5) Third Strand Synthesis

Samples No.2, No.3 and No.4 were processed further by resuspension ofthe beads in 26 ul of 10 mM Tris-HCl (pH 7.5) and heating at 90° C. for3 minutes. The second strands released by this process were isolatedapart from the beads by magnetic separation and mixed with 40 pMoles of3^(rd) strand primers for a final volume of 30 ul. For Sample No.3, the3^(rd) strand primers were T7-T₂₅ primers with the sequence 5′GGCCAGTGAATTGTAATACGACTCACTATAGGGATC(T)₂₅-3′

For Samples No.2 and No.4, the 3^(rd) strand primers were T3-T₂₅ primerswith the sequence: 5′ CTCAACGCCACCTAATTACCCTCACTAAAGGGAGAT(T)₂₅-3′

After mixing, Samples No.2, No.3 and No.4 were kept on ice for 15minutes. Extension reactions were then set up in 1× M-MuLV Buffer (NewEngland Biolabs, Beverly Mass.) with 10 units of M-MuLV ReverseTranscriptase (New England Biolabs, Beverly Mass.) and 1 mM of each dNTPin a final volume of 40 ul. Incubation was carried out for one hour at37° C. 6 units of T4 DNA Polymerase (New England Biolabs, Beverly,Mass.) were added to Samples No.1, No.2, No.3 and No.4 and incubationcarried out for a further 15 minutes at 37° C. Reactions were stopped bythe addition of EDTA (pH 8.0) to a final concentration of 10 mM. The DNAfrom Samples 2, No.3 and No.4 was then purified by adjusting the volumesto 150 ul by adding appropriate amounts of 10 mM Tris-HCl. Reactionswere mixed with an equal volume of Phenol:chloroform:isoamyl alcohol(25:24:1) and transferred to 2 ml Phase Lock Gel Heavy tubes (Eppendorf,Westbury, N.Y.). Tubes were vorteed for 1-2 minutes and centrifuged for10 minutes at 16,000 rpm in a microfuge. The aquaeous phase was thentransferred to another tube and DNA precipitated with Ethanol andAmmonium Acetate.

6) Transcription

Beads (Sample No.1) and precipitates (Samples No.2, No.3 and No.4) wereresuspended with components from the BioArray High. Yield TranscriptionKit (T7) (ENZO Diagnostics, N.Y.) and transcription carried out in a 20ul volume following the manufacturer's directions with the addition of 5uCi ³H-CTP, 20 Ci/mMol (Amersham Pharmacia Biotech, Piscataway, N.J.).In addition some reactions were carried out as described above, but T3RNA polymerase from the BioArray High Yield Transcription Kit (T3) (ENZODiagnostics, N.Y.) was substituted. Reactions were carried out for 120minutes at 37° C.

7) Results 2^(nd) strand 3^(rd) strand Sample No. Primer Primer RNAPolym CPM No. 1 T7-C9 — T7 12,392 No. 2 T7-C9 T3-T₂₅ T7 29,160 No. 2T7-C9 T3-T₂₅ T3 14,784 No. 3 C9 T7-T₂₅ T7 22,622 No. 4 C9 T3-T₂₅ T312,2218) Conclusions

This example demonstrated that a promoter can be introduced during3^(rd) strand synthesis to create functional production centers. Thisexample also demonstrated that in addition to a T7 promoter, a T3promoter was also functional in the present method. This example alsodemonstrated that different production centers could be introduced intoeach end of a construct (Sample No.2) and both production centers werefunctional.

EXAMPLE 9 Multiple Rounds of 2^(nd) Strand Synthesis with ThermostablePolymerases

1) Preparation of Beads, Binding of RNA to Beads and First strandsynthesis were carried out as described in Example 8.

2) Second Strand Synthesis and Recycling

Poly dG addition was carried out as described for sample No. 3 inExample 6 and the beads with tailed 3′ ends were used for 2^(nd) strandsynthesis under various conditions. 50 ul Reactions mixes were set up asfollows: Sample No.1 consisted of 1× Taq PCR Buffer (Epicentre, Madison,Wis.), 3 mM MgCl₂, 1×PCR Enhancer (Epicentre, Madison, Wis.), 0.4 mMdNTPs, 40 pMoles C9 primers and 5 units of Master Amp™ Taq DNAPolymerase (Epicentre, Madison, Wis.); Sample No.2 was the same assample No.1 except 100 pMoles of C9 primers were used; Sample No.3consisted of 1× Tth PCR Buffer (Epicentre, Madison, Wis.), 3 mM MgCl₂,1× PCR Enhancer (Epicentre, Madison, Wis.), 0.4 mM dNTPs, 40 pMoles C9primers and 5 units of Master Amp™ Tth DNA Polymerase (Epicentre,Madison, Wis.); Sample No.4 was the same as sample No.3 except 100pMoles of C9 primers were used Samples No.1 and No.3 went through onebinding/extension cycle while samples No.2 and No.4 went through 5 suchcycles. Each binding extension/extension cycle was carried out in athermocycler under the following conditions:

-   -   2 minutes at 90° C.    -   5 minutes at 4° C.    -   5 minutes at 37° C.    -   5 minutes at 50° C.    -   15 minutes at 72° C.

At the end of each cycle, samples No.2 and No.4 were briefly shaken toresuspend the beads. After the completion of either 1 or 5 cycles, themixtures were heated at 90° C. for 3 minutes and the aqueous portioncollected after magnetic separation. Each sample was phenol extractedand ethanol precipitated as described previously in step 5 of Example 8for samples No.3 and No.4.

3) Third Strand Synthesis

Pellets were resuspended in 26 ul of 10 mM Tris-HCl (pH 7.5) and T7-T25primers were added. For Samples No.1 and No.3, 40 pMoles of T7-T₂₅ wereadded; for Samples No.2 and No.4, 400 pMoles of T7-T25 were added. Thirdstrand synthesis was then carried out by the addition of MuLV, MuLVbuffer and dNTPS as described in step 5 of Example 8.

4) Transcription

Transcription was carried out as described previously without theaddition of radioactive precursors. Analysis of the reaction from eachsample was carried out by gel electophoreis as described previously andshown in FIG. 31.

5) Conclusions

This example demonstrated that thermostable polymerases could be usedfor 2^(nd) strand synthesis in the methods described above. This examplealso demonstrated that by increasing the amount of primers and thenumber of cycles the amount of RNA copies derived from the originallibrary of nucleic acids was increased.

EXAMPLE 10 Levels of Transcription Derived from Sequential Rounds of2^(nd) Strand Synthesis

1) Preparation of Beads, Binding of RNA to Beads and First strandsynthesis were carried out as described in Example 8 except the amountof analytes and reagents for each reaction was increased two-fold.Preparation of 1^(st) strands for 2^(nd) strand synthesis was carriedout as described previously for sample 3 in Example 6.

2) Second Strand Synthesis

Second strand synthesis was carried out as described for Sample No.3 inExample 8. Separation and isolation of the 2^(nd) strand products wascarried out as described in Example 8 and set aside as Sample No. 1.Fresh reagents were then added to the beads and another round of 2^(nd)strand synthesis was carried out. The products of this second reactionwere separated from the beads and designated Sample No.2. The beads werethen used once more for a third round of synthesis. The products of thisreaction were set aside as Sample No.3.

3) Third Strand Synthesis

Samples No.1, No.2 and No.3 were used as templates for 3^(rd) strandsynthesis in individual reactions with the reagents and conditionpreviously described in Example 8. As mentioned above, the startingmaterial in the present example was twice the amount used in example 8and as such the amounts of all reagents were doubled for this reactionas well. For example, 80 pMoles of T7-T25 primers were used.Purification of the products from each reaction was carried out asdescribed in Example 8.

4) Transcription

Transcription reactions were carried out as with the BioArray High YieldTranscription Kit (T7) (ENZO Diagnostics, NY). The DNA was used in a 20ul final reaction volume which was incubated for 2 hours at 37° C. Gelanalysis was then used to evaluate the amount of synthesis that was aresult of each round of 2^(nd) strand synthesis described above. Forpurposes of contrast, various amounts of the transcription reaction (4ul and 10 ul) were analyzed and in addition equvalent amounts of the DNAtemplate that were not used in transcription reactions were alsoincluded. The results of this are shown in FIG. 32.

5) Conclusion

This example demonstrated that the 2^(nd) strands made in each round of2^(nd) strand synthesis were substantially equal in their ability to beused to synthesize a library with functional production centers. FIG. 32also shows the contrast between the amount of transcript and theoriginal DNA templates used for this synthesis thereby demonstrating thehigh levels of synthesis from each template.

Example 11 Use of Reverse Transcriptases from Various Sources

Preparation of Beads, Binding of RNA to Beads and 1^(st) strandsynthesis were carried out as described in Example 6 except that ReverseTranscriptases from various sources were used for 1^(st) strandsynthesis reactions. 2^(nd) strand synthesis was carried out asdescribed in Example 6 for sample No.2, i.e Terminal Transferaseaddition followed by binding and extension of T7-C9 primers. A list ofthe various Reverse Transcriptases and their sources is given below.

-   1) Superscript II [RNaseH(−) MuLV] (Life Technologies, Rockville,    Md.)-   2) RNase H(+) MuLV (Life Technologies, Rockville, Md.)-   3) RNase H(+) MuLV (New England Biolabs, Beverly, Mass.)-   4) Enhanced AMV (Sigma, St. Louis, Mo.)-   5) AMV (Life Technologies, Rockville, Md.)-   6) AMV (Sigma, St. Louis, Mo.)-   7) Omniscript (Qiagen-   8) Display THERMO-RT Display Systems Biotech,-   9) Powerscript [RNaseH(−) MuLV] (Clontech laboratories,

Each 2^(nd) stand synthesis was carried out in the buffer provided bythe manufacturer for each Reverse Transcriptase with the exception ofthe New England Biolabs version of RNase H (+) MuLV which was used inthe buffer provided for the Life Technologies version of RNase H (+)MuLV. Further processing and transcription reactions were as previouslydescribed in Example 6. The results of this experiment re shown in FIG.33.

Conclusions

A variety of different Reverse Transcriptases ccould be used inconjunction with the methods of the present invention.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art in light of the above detailed description and examplesof the present invention. All such variations are fully embraced by thescope and spirit of the invention as more particularly defined in theclaims that now follow.

1. (canceled) 2-784. (canceled)
 785. A composition of matter thatcomprises an array of solid surfaces comprising discrete areas; whereinat least two of said discrete areas each comprises: a first set ofnucleic acid primers; and a second set of nucleic acid primers; whereinthe nucleotide sequences in said first set of nucleic acid primers aredifferent from the nucleotide sequences in said second set of nucleicacid primers; wherein the nucleotide sequences of a first set of nucleicacid primers of a first discrete area and the nucleotide sequences of afirst set of nucleic acid primers of a second discrete area differ fromeach other by at least one base; and wherein the nucleotide sequences ofthe second set of nucleic acid primers of a first discrete area and thenucleotide sequences of the second set of nucleic acid primers of asecond discrete area are substantially the same or identical.
 786. Thecomposition of claim 785, wherein said array of solid surfaces has beendesigned/synthesized such that D1 is less than D2, said D1 being thephysical distance on said array between a nucleic acid primer that ispart of a first set of an area and the nucleic acid primer is part of asecond set of the same area, and D2 being the physical distance in anucleic acid in a sample between the sequence of a primer binding sitein said nucleic acid in a sample for the nucleic acid primer of thefirst set and the complement of the primer binding site in the saidnucleic acid in the sample for the nucleic acid primer in the secondset.
 787. The composition of claim 785, wherein said nucleic acidprimers are selected from the group consisting of DNA, RNA and analogsthereof.
 788. The composition of claim 787, wherein said analogscomprise PNA.
 789. The composition of claims 787 or 788, wherein saidnucleic acids or analogs are modified on any one of the sugar, phosphateor base moieties.
 790. The composition of claim 785, wherein said solidsurfaces are porous or non-porous.
 791. The composition of claim 789,wherein said porous solid surfaces are selected from the groupconsisting of polyacrylamide and agarose.
 792. The composition of claim790, wherein said non-porous solid surfaces comprise glass or plastic.793. The composition of claim 785, wherein said solid surfaces aretransparent, translucent, opaque or reflective.
 794. The composition ofclaim 785, wherein nucleic acid primers are directly or indirectly fixedor immobilized to said solid surfaces.
 795. The composition of claim794, wherein said nucleic acid primers are indirectly fixed orimmobilized to said solid surfaces by means of a chemical linker orlinkage arm.
 796. A composition of matter that comprises an array ofsolid surfaces comprising a plurality of discrete areas; wherein atleast two of said discrete areas each comprises: a first set of nucleicacid primers; and a second set of nucleic acid primers; wherein thenucleotide sequences in said first set of nucleic acid primers aredifferent from the nucleotide sequences in said second set of nucleicacid primers; wherein the nucleotide sequences of a first set of nucleicacid primers of a first discrete area and the nucleotide sequences of afirst set of nucleic acid primers of a second discrete area differsubstantially from each other; and wherein the nucleotide sequences ofthe second set of nucleic acid primers of a first discrete area and thenucleotide sequences of the second set of nucleic acid primers of asecond discrete area are substantially the same or identical.
 797. Thecomposition of claim 795, wherein said array of solid surfaces has beendesigned/synthesized such that D1 is less than D2, said D1 being thephysical distance on said array between a nucleic acid primer that ispart of a first set of an area and the nucleic acid primer is part of asecond set of the same area, and D2 being the physical distance in anucleic acid in a sample between the sequence of a primer binding sitein said nucleic acid in a sample for the nucleic acid primer of thefirst set and the complement of the primer binding site in the saidnucleic acid in the sample for the nucleic acid primer in the secondset.
 798. The composition of claim 796, wherein said nucleic acidprimers are selected from the group consisting of DNA, RNA and analogsthereof.
 799. The composition of claim 798, wherein said analogscomprise PNA.
 800. The composition of claims 798 or 799, wherein saidnucleic acids or analogs are modified on any one of the sugar, phosphateor base moieties.
 801. The composition of claim 796, wherein said solidsurfaces are porous or non-porous.
 802. The composition of claim 801,wherein said porous solid surfaces are selected from the groupconsisting of polyacrylamide and agarose.
 803. The composition of claim796, wherein said non-porous solid surfaces comprise glass or plastic.804. The composition of claim 796, wherein said solid surfaces aretransparent, translucent, opaque or reflective.
 805. The composition ofclaim 796, wherein nucleic acid primers are directly or indirectly fixedor immobilized to said solid surfaces.
 806. The composition of claim805, wherein said nucleic acid primers are indirectly fixed orimmobilized to said solid surfacees by means of a chemical linker orlinkage arm. 807-952. (canceled)